Tol1 FACTOR TRANSPOSASE AND DNA INTRODUCTION SYSTEM USING THE SAME

ABSTRACT

An object is to provide a Tol1 element transposase and a use thereof. Provided is a Tol1 element transposase containing (a) a protein having the amino acid sequence of SEQ ID No: 1 or (b) a protein having an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 1 and having an enzymatic activity for transferring Tol1 element. Further, provided are a polynucleotide encoding the transposase and an expression construct containing the polynucleotide therein. The present invention also provides a DNA introduction system including (a) a donor factor having such a structure that a desired DNA is inserted in a transposase gene-defected Tol1 element and (b) a helper factor containing the transposase or the polynucleotide.

TECHNICAL FIELD

The present invention relates to an enzyme for catalyzing transpositionof a transposon (hereinafter referred to as a transposase) and a usethereof. Specifically, the present invention relates to a transposase ofTol1 element (Transposable element of Oryzias latipes, no. 1) that is atransposon derived from medaka fish and a polynucleotide encoding thetransposase, a DNA introduction system and a DNA introduction methodusing the transposase, and a DNA introducing kit for use in the systemand the like.

BACKGROUND ART

A DNA transposable element is one kind of repeat sequences structuringbiological genomes, and present in vertebrate genomes in a large amount.However, most of vertebrate DNA transposable elements lose transposableactivities to be debris. Vertebrate DNA transposable elements whosetransposition was directly demonstrated are only a zebrafish Tzf element(Lam W L, Lee T S, Gilbert W. (1996) Proc Natl Acad Sci USA 93:10870-10875) and a medaka fish Tol2 element (Transposable element ofOryzias latipes, no. 2) (Koga A., Suzuki M., Inagaki H., Bessho Y. andHori H. (1996) Transposable element in fish. Nature 383: 30).

The Tol1 element is a DNA element present in medaka fish genomes in 100to 200 copies (Koga A., Sakaizumi M., Hori H. (2002) Zoolog Sci 19: 1 to6 (Non-patent Document 1)). This element was discovered as a pieceinserted in a tyrosinase gene of a mutant showing a complete albino bodycolor (Koga A., Inagaki H., Bessho Y., and Hori H. (1995) Mol Gen Genet249: 400-405. (Non-patent Document 2)). Tyrosinase is an enzymeessential to biosynthesis of melanin. A transposition activity waseasily demonstrated in Tol2 discovered thereafter. Different from Tol2,since excision and insertion was not directly detected in the Tol1element, it was originally considered that the Tol1 element was anelement that had already lost a transposition activity. In addition tothe copy first discovered in a tyrosinase gene, other copies were alsoisolated to be examined, but a structure that was estimated to be a genewas not found (Koga A., Inagaki H., Bessho Y., and Hori H. (1995) MolGen Genet 249: 400-405. (Non-patent Document 2)). This fact also servesas a reason that Tol1 was considered to be an element that had alreadylost a transposition activity.

In 2001, an organism having partial pigmentation in the body, that is, amosaic pigmented organism was found as one albino subline. As a resultof analysis on this organism, it was demonstrated that the Tol1 elementwas a body cell and dropped out of its insertion site (Tsutsumi M., ImaiS., Kyono-Hamaguchi Y., Hamaguchi S., Koga A. and Hori H. (2006) PigmentCell Res 19: 243-247. (Non-patent Document 3)). Occurrence of thephenomenon of dropping means that Tol1 is a DNA transposable elementthat has not lost a transposition activity. However, de novo insertionof this element in a chromosome has never been observed. Furthermore, notransfer enzyme (transposase) has been found.

By the way, transposable elements are utilized in a genetic engineeringtechnique or a molecular technique. For example, utilization andapplication of transposable elements to trapping of mutagenesis, genes,promoters, enhancers, etc., gene therapies, and the like are expected.The Tol2 element, which was discovered as an element present in medakafish genomes, has already been provided for such applications (Koga A.,Hori H., and Sakaizumi M. (2002) Mar Biotechnol 4: 6-11. (Non-patentDocument 4), Johnson Hamlet M. R., Yergeau D. A., Kuliyev E., Takeda M.,Taira M., kawakami K., Mead P. E. (2006) Genesis 44: 438-445.(Non-patent Document 5), Choo B. G., Kondrichin I., Parinov S.,Emelyanov A., Go W., Toh W. C. and Korzh V. (2006) BMC Dev Biol 6: 5.(Non-patent Document 6), Japanese Patent Application Laid-Open (JP-A)No. 2001-218588 (Patent Document 1)). In addition to the Tol2 element,Sleeping Beauty element artificially reconstructed from debris presentin salmon genomes (Lvics Z., Hackett P. B., Plasterk R. H., Izsvak Z.(1997) Cell 91: 501-510. (Non-patent Document 7), National Publicationof International Patent Application No. 2001-523450 (Patent Document2)), Frog Prince element reconstructed from a frog in the same manner(Miskey C., Izsvak Z., Plasterk R. H., Ivics Z. (2003) Nucleic Acids res31: 6873-6881. (Non-patent Document 8), National Publication ofInternational Patent Application No. 2005-527216 (Patent Document 3)),and piggyBac element isolated from an insect (Wu S. C., Meir Y. J.,Coates C. J., Handler A. M., Pelczar P., Moisyadi S, and Kaminski J. M.(2006) Proc Natl Acad Sci USA 103: 15008-15013. (Non-patent Document 9))have been used in gene introduction, etc. These elements have acharacteristic of high transposition frequency. This characteristic issignificantly important when considering utilization and application togenetic engineering techniques or molecular biological techniques. Alsowith respect to Tol1, it is estimated that transposition frequencythereof is high since the number of pigmented cells is large in medakafish discovered by the present inventors.

Patent Document 1: JP-A No. 2001-218588 Patent Document 2: NationalPublication of International Patent Application No. 2001-523450 PatentDocument 3: National Publication of International Patent Application No.2005-527216

[Non-patent Document 1] Koga A., Sakaizumi M., Hori H. (2002) Zoolog Sci19: 1-6.

[Non-patent Document 2] Koga A., Inagaki H., Bessho Y., Hori H. (1995)Mol Gen Genet 249: 400-405.

[Non-patent Document 3] Tsutsumi M., Imai S., Kyono-Hamaguchi Y.,Hamaguchi S., Koga A., Hori H. (2006) Pigment Cell Res 19: 243-247.

[Non-patent Document 4] Koga A., Hori H., Sakaizumi M. (2002) MarBiotechnol 4: 6-11.

[Non-patent Document 5] Johnson Hamlet M. R., Yergeau D. A., Kuliyev E.,Takeda M., Taira M., Kawakami K., Mead P. E. (2006) Genesis 44: 438-445.

[Non-patent Document 6] Choo B. G., Kondrichin I., Parinov S., EmelyanovA., Go W., Toh W. C., Korzh V. (2006) BMC Dev Biol 6: 5.

[Non-patent Document 7] Ivics Z., Hackett P. B., Plasterk R. H., IzsvakZ. (1997) Cell 91: 501-510.

[Non-patent Document 8] Miskey C., Izsvak Z., Plasterk R. H., Ivics Z.(2003) Nucleic Acids Res 31: 6873-6881.

[Non-patent Document 9] Wu S. C., Meir Y. J., Coates C. J., Handler A.M., Pelczar P., Moisyadi S., Kaminski J. M. (2006) Proc Natl Acad SciUSA 103: 15008-15013.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Tol1 element is largely expected in terms of unitization and applicationas a novel transcription factor, but all Tol1 elements discovered so farare copies with internal deletion, and transposase and a gene thereofthat are considered to be contained in a full-length copy have not beenidentified.

In order to utilize a transposable element in a genetic engineeringtechnique or a molecular biological technique, a transposase that is afactor transferring the element is required in addition to an element tobe a vector.

An object of the present invention is thus to provide a Tol1 elementtransposase in order to use Tol1 element in a genetic engineeringtechnique, etc. Another object of the present invention is to provideuses of Tol1 element (such as a DNA introduction system and a DNAintroduction method).

Means for Solving the Problems

The present inventors tried to identify a transposase gene of Tol1element using the above-described mosaic pigmented organism (medakafish) as an experimental material. Database search was first repeatedlyperformed, thereby constructing a sequence that is estimated to be abase sequence of a transposase gene. Subsequently, the inventorssucceeded in identification of 2.9 kb of cDNA, through analysis on mRNAof the mosaic pigmented organism. As a result of examining the sequenceof this cDNA, it was revealed that a sequence corresponding to 851 aminoacids was present in its inside. On the other hand, it was confirmedthat a protein encoded by this cDNA caused transposition of Tol1 elementin both human and mouse cells. Further, as a result of examination of atransposition frequency, a high value comparable to that of Tol2 wasshown. The fact means that Tol1 element has the same utility value and apossibility as Tol2 element as a transposon. The Tol1 element has a highutility value also in respect of capability to serve as an alternativemeans to Tol2 element. That is, the Tol1 element has a possibility toeffectively act on cell lines and organism species in which sufficienttransposition frequencies cannot be obtained by Tol2 element.

As a result of further studies, the noticeable finding that Tol1 andTol2 never induce mutual transposition (that is, a Tol1 elementtransposase does not induce transposition of Tol2, and vice versa) wasobtained. Based on this finding, it would be possible to introduce twotypes of DNAs successively to a target cell utilizing both Tol1 elementand Tol2 element. Further, after introducing the two types of DNAs insuch a manner, supplying a transposase corresponding to one of theelements allows only one of the DNAs to be specifically transferred.Accordingly, the fact that Tol1 element and Tol2 element do not give aninfluence on each other's transposition exceptionally enhancesusefulness and utility value of Tol1 element.

As a result of further advanced studies, existence of Tol1 elementhaving a length such as 18 kb and 20 kb was revealed. This fact suggeststhat Tol1 element is useful as a means for introducing (transporting) aDNA fragment with a large size. Two kinds of experiments were performedfocusing on this point. For the first experiment, removal of an internalregion unnecessary for transposition was tried in the Tol1 element(Tol1-tyr, 1855 base pairs, SEQ ID NO: 10), which had been discovered asa fragment inserted in a tyrosinase gene. As a result, it was revealedthat Tol1 element was transported without damaging a transpositionefficiency thereof only if 157 by of the left end (5′ end region) and106 by of the right end (3′ end region) are present. For the secondexperiment, transposition efficiencies were measured in the case ofinserting DNA fragments with various sizes in short Tol1 element havinga deleted internal region as described above. As a result, as the sizeof a DNA fragment to be inserted was larger (i.e. as the distancebetween the left end and the right end of Tol1 element was longer), thetransposition frequency was lower. However, even when the size of a DNAfragment to be inserted was maximum and the distance between the leftend and the right end of Tol1 element was 22.1 kb, the transpositionfrequency thereof was still significantly higher than a frequency ofrandom incorporation into chromosomes without employing transposition.Although a plurality of DNA transposable elements used in mammals exist,an element having a length of 22.1 kb is the longest among the elementsreported so far. As described above, as a result of the studies made bythe inventors, Tol1 1 element was demonstrated to be excellent in itsloading ability and exceptionally useful for a means for introducing(transporting) a DNA fragment with a large size.

As a result of further studies, it was revealed that excision of Tol1element occurred also in Xenopus laevis that is important as a model forstudies of genetics and development of vertebrates, and it was suggestedthat Tol1 element functioned as a transposable element also in cells ofXenopus laevis. This suggestion could be an important finding thatsupports greatness of versatility of Tol1 element.

On the other hand, it was confirmed that Tol1 element functioned also ininsects. First, a donor plasmid containing a nonautonomous copy of Tol1element was injected into a fertilized egg of a silkworm together withRNA encoding a transfer enzyme of Tol1 element. The fertilized egg waskept warm to promote development, and a plasmid DNA was then recoveredfrom the embryo. Subsequently, the structure was analyzed by PCR. As aresult, it was found that the structure had molecules in which portionsof Tol1 element had been drawn out. Further, a genomic DNA was extractedfrom the embryo and analyzed by a technique of inverse PCR. The analysisrevealed that Tol1 element had been incorporated into chromosomes. Asdescribed above, both excision and insertion that are two stages of atransfer reaction occurred in the silkworm. This result has thefollowing three meanings: (1) a transfer reaction of Tol1 element doesnot require an element from a host organism, or if the element isrequired, it is commonly owned by biological species of protostomes anddeuterostomes; (2) systems of gene introduction, gene trapping,mutagenesis, and the like, which can be used in silkworms, can beconstructed utilizing Tol1 element; and (3) similar systems capable ofbeing used in wide varieties of animals can be constructed.

As described above, not only a Tol1 element transposase was successfullyidentified, it was also revealed that Tol1 element had preferableproperties for a transposable element for use in genetic engineeringtechniques. The present invention is based on such achievements andprovides the following transposase, DNA introduction system, and thelike.

[1] A Tol1 element transposase containing any of proteins selected fromthe group consisting of the following (a) to (c):

(a) a protein having an amino acid sequence encoded by the base sequenceof SEQ ID NO: 1;

(b) a protein having the amino acid sequence of SEQ ID NO: 2; and

(c) a protein having an amino acid sequence homologous to the amino acidsequence of SEQ ID NO: 2, and having an enzymatic activity fortransferring Tol1 element.

[2] A polynucleotide encoding a Tol1 element transposase containing anyof base sequences selected from the group consisting of the following(a) to (c):

(a) a base sequence encoding the amino acid sequence of SEQ ID NO: 2;

(b) the base sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4;and

(c) a base sequence homologous to the base sequence (b) and encoding aprotein having an enzymatic activity for transferring Tol1 element.

[3] An expression construct containing the polynucleotide according to[2].

[4] The expression construct according to [3], further containing apromoter operably linked to the polynucleotide.

[5] The expression construct according to [3] or [4], further containinga poly-A additional signal sequence or a poly-A sequence connected tothe polynucleotide in the downstream side.

[6] A DNA introduction system including:

(a) a donor factor having such a structure that a desired DNA isinserted in a transposase gene-defected Tol1 element; and

(b) a helper factor containing the transposase according to [1] or thepolynucleotide according to [2].

[7] The DNA introduction system according to [6], wherein the Tol1element has the inverted repeat sequence of SEQ ID NO: 5 in the 5′ endregion and the inverted repeat sequence of SEQ ID NO: 6 in the 3′ endregion.

[8] The DNA introduction system according to [6], wherein the Tol1element contains DNA of the following (a) or (b):

(a) DNA having the base sequence of any of SEQ ID NOs: 10 to 12; or

(b) DNA having a base sequence homologous to the base sequence of any ofSEQ ID NOs: 10 to 12, wherein a transposase having the amino acidsequence of SEQ ID NO: 1 binds to its end.

[9] The DNA introduction system according to [6], wherein the Tol1element contains 5′ end side DNA and 3′ end side DNA obtained bydeleting at least from the 158th base to the 1749th base counting fromthe 5′ end in the base sequence of SEQ ID NO: 10.

[10] The DNA introduction system according to [6], wherein the Tol1element contains DNA having the base sequence of SEQ ID NO: 21 and DNAhaving the base sequence of SEQ ID NO: 22.

[11] The DNA introduction system according to any of [8] to [10],wherein a target site duplicated sequence is connected to the 5′ end andthe 3′ end of the toll element.

[12] The DNA introduction system according to [11], wherein the targetsite duplicated sequence contains the sequence of any of SEQ ID NOs: 13to 15.

[13] The DNA introduction system according to any of [6] to [12],wherein the desired DNA is a gene.

[14] The DNA introduction system according to any of [6] to [13],wherein the donor factor is a vector obtained by inserting a desired DNAin a transposase gene-defected Tol1 element, and the helper factor is avector containing the polynucleotide according to [2].

[15] The DNA introduction system according to [14], wherein the vectorbeing the helper factor further contains a promoter operably linked tothe polynucleotide.

[16] The DNA introduction system according to [14] or [15], wherein thevector being the helper factor further contains a poly-A additionalsignal sequence or a poly-A sequence connected to the polynucleotide inthe downstream side.

[17] A DNA introduction method including a step of introducing the DNAintroduction system according to any of [6] to [16] to a target cellwhich is a vertebrate cell.

[18] The DNA introduction method according to [17], wherein the targetcell is a vertebrate cell other than a cell in a state of a constituentfactor of a human individual.

[19] The DNA introduction method according to [17] or [18], furtherincluding a step of introducing DNA different from the desired DNAintroduced by the DNA introduction system to the target cell byutilizing Tol2 element.

[20] A method of transferring a specific DNA site on genomic DNA,including a step of supplying a transposase corresponding to Tol1element or Tol2 element to a cell genetically manipulated with the DNAintroduction method according to [19].

[21] A method of transferring a specific DNA site on genomic DNA,including a step of introducing the transposase according to [1] or thepolynucleotide according to [2] into a cell having a transposasegene-defected Tol1 element on genomic DNA.

[22] The method according to [21], wherein another polynucleotidesequence is inserted in the Tol1 element.

[23] A cell genetically manipulated by the DNA introduction systemaccording to any of [6] to [16], the DNA introduction method accordingto any of [17] to [19], or the method according to any of [20] to [22].

[24] A DNA introducing kit, including a donor factor made of anexpression construct containing a transposase gene-defected Tol1 elementand an insertion site, and a helper factor made of an expressionconstruct containing the transposase according to [1] or thepolynucleotide according to [2].

[25] The DNA introducing kit according to [24], wherein the toll elementhas a structure having the insertion site between 5′ end side DNA and 3′end side DNA, which is obtained by deleting at least from the 158th baseto the 1749th base counting from the 5′ end in the base sequence of SEQID NO: 10.

[26] The DNA introducing kit according to [24], wherein the Tol1 elementhas a structure having the insertion site between DNA having the basesequence of SEQ ID NO: 21 and DNA having the base sequence of SEQ ID NO:22.

[27] The DNA introducing kit according to any of [24] to [26], whereinthe insertion site is made of a plurality of different kinds ofrestriction enzyme recognition sites.

[28] The DNA introducing kit according to any of [24] to [27], whereinthe donor factor is a vector containing a transposase gene-defected Tol1element and an insertion site, and the helper factor is a vectorcontaining the polynucleotide according to [2].

[29] The DNA introducing kit according to [28], wherein the vector beingthe helper factor further contains a promoter operably linked to thepolynucleotide.

[30] The DNA introducing kit according to [28] or [29], wherein thevector being the helper factor further contains a poly-A additionalsignal sequence or a poly-A sequence connected to the polynucleotide inthe downstream side.

[31] A reconstructed transposon having a structure inserted with thepolynucleotide according to [2] in a transposase gene-defected Tol1element.

[32] The transposon according to [31], containing a promoter operablylinked to the polynucleotide.

[33] The transposon according to [31] or [32], containing a poly-Aadditional signal sequence or a poly-A sequence connected to thepolynucleotide in the downstream side.

[34] A DNA introduction system, containing the transposon according toany of [31] to [33].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a mosaic pigmented fish. Panels A to C show the same fish.A fish having a black eye on one side and a red eye on the other sidewas photographed from the right side (panel A), the front (panel B), andthe left side (panel C). The fish in Panel D is pigmented in a widerange of the eyes and has a large number of pigmented dots on the backskin. The dots on the back were indicated with triangles. Panel E showsan organism having spoke-form pigmentation in an eye. Panel F shows aperitoneum. A peritoneum is densely pigmented in wild fish, andpigmentation is not recognized in albino fish.

FIG. 2 shows structures of Tol1 nonautonomous and autonomous copies.Tol1-tyr is the first discovered Tol1 nonautonomous copy, and wasinserted in a tyrosinase gene of fish A. Tol1-L1 is the completeautonomous copy successfully identified in this time, and has afunctional transposase gene in its inside. Genomic DNA of fish B wassheared and fragments with 36 to 48 kb were taken out and inserted infosmid vector pCC1FOS to form a genomic library. This library wasscreened to obtain Tol1-L1. A transposase gene (exon) in its inside isshown in a bar. An initiation codon (ATG) and a termination codon (TAG)of this gene are also shown. The underlined parts noted by a to e wereused for forming probes of hybridization. x and y indicate positions ofa primer of 3′ RACE and a primer of 5′ RACE, respectively. Sequences ofthese primers (primer x: SEQ ID NO: 16, primer y: SEQ ID NO: 17)correspond to positions of bases 152 to 181 and 457 to 332 having thebase sequence (SEQ ID NO: 3) registered in DDBJ/EMBL/GenBank asAccession No. AB264112, respectively.

FIG. 3 shows results of the RACE analysis. RNA was extracted fromembryos after 7 days from fertilization of fish A and fish B andsynthesis of a cDNA single strand was performed from an oligo-dT primer.Then, using primer x and a 3′ adaptor primer contained in a RACE kit, 3′RACE of this cDNA single strand was performed. A PCR product waselectrophoresed with a 1.0% agarose gel and transcribed in a nylon film,and then hybridization with the probe b was performed. The left panelshows a picture taken immediately after the electrophoresis and theright panel show a picture showing the result of the hybridization. Itwas found that one band as a signal appeared only in fish B.Subsequently, a portion corresponding to a signal of a gel was excisedto recover a DNA fragment contained therein, the DNA fragment wasconnected to a plasmid vector and a clone to which the probe bound wasisolated by colony hybridization. 5′ RACE was performed only on RNA offish B. The primers used were y and a 5′ adaptor primer contained in thekit. Operations after PCR were the same as in the case of 3′RACE exceptthat the probe e was used in hybridization. One band appeared as aresult of the hybridization and isolated by the same method.

FIG. 4 shows results of checking of a Tol1 element transposase and apart of other transposase of the hAT family by the Clustal X program. Ithas been known that an element of the hAT family has some regions whereamino acid sequences are preserved. The regions are expressed by A to F(Cited document 27). D and F out of A to F are located in acomparatively short region and are considered to be associated withdimerization of a protein. Elements having high homology to Tol1 wereselected from the elements registered as the hAT family members (GenBank Accession No. PF05699,http://www.ncbi.nlm.nih.gov/Genbank/index.html) from such a viewpointthat various host biological species were contained and the sequence waschecked. Results thereof are shown in FIG. 4. Names encoded in UniProtKBwere used as names of respective elements. Five letters indicating hostbiological species are added to the names. In addition, portions ofamino acids used for checking are expressed by the numbers that indicatepositions thereof. A Clustal X default method was directly employed forcoloration of the amino acids.

FIG. 5 shows a southern blot analysis of copies of Tol1 contained inmedaka fish genomes. Genomic DNA was extracted from each one of fish A,fish B, HNI and Hd-rR. HNI and Hd-rR are inbred lines that arefrequently used in studies on medaka fish. 8.0 μg of genomic DNA wasprepared for each fish, completely excised with restriction enzyme PvuIIand electrophoresed with a 1.0% agarose gel to be transcribed on a nylonfilm, which was then subjected to hybridization with probes a to d (seeFIG. 2 for positions). Positions of DNA fragments known in sizes(molecular weight markers) are shown in the left side of the pictures.It was found from the results shown herein that internal deletion wasrecognized in most copies of Tol1 present in medaka fish genomes.

FIG. 6 shows a plasmid used in transposition frequency measurement.Tol1-tyr (GenBank Accession No. D42062, SEQ ID NO: 10) was amplifiedfrom genomic DNA of fish A with 8 bp of adjacent TSD (CCTTTAGC (SEQ IDNO: 13)) and inserted in plasmid pUC19 to form a clone. Subsequently, apart of plasmid pCMV-Tag1 (bases 1675 to 3474 of the base sequence ofGenBank Accession No. AF025668, SEQ ID NO: 18) was amplified by PCR andinserted in a SalI recognition site (bases 706 to 711 of the basesequence of GenBank Accession No. D42062) that is present in one site ofTol1-tyr. A neomycin-resistant gene is contained in this part ofpCMV-Tag1. The thus prepared plasmid was used as a donor. A helper wasprepared by inserting bases 31 to 2817 (SEQ ID NO: 19) of Tol1 cDNA(base sequence of GenBank Accession No. AB264112, SEQ ID NO: 3) in amulticloning site of plasmid pCI. This multicloning site was presentbetween a CMV promoter and a poly-A additional signal. A defectivehelper was prepared by adding modification to a base sequence of ahelper with PCR. The bases 996 to 1001 of the helper are ATGAAA, whichcorresponds to amino acids, methionine and lysine. This sequence wasaltered into TAGTAA. This alteration resulted in sequential generationof two termination codons in about the middle of ORF of a transposase. Afiller plasmid was prepared by inserting 2.8 kb of a DNA fragment inplasmid pCI in place of transposase cDNA.

FIG. 7 shows transposition of Tol1 in mammal cells. A donor and ahelper, a donor and a defective helper, or only a donor was incorporatedin HeLa cells and NIH/3T3 cells. However, fillers were added asnecessary. Selection in G418 was then carried out. A picture of 60mm-dish dyed with a Giemsa stain solution is shown. A large number ofG418-resistant colonies were generated only in the case where a donorand a helper were incorporated.

FIG. 8 shows base sequences of insertion points of inserted Tol1 copies.Genomic DNA was extracted from G418-resistant cells obtained byintroducing a donor and a helper and cut with EcoRI or PstI. The tworestriction enzymes do not cut the donor. Subsequently, after thegenomic DNA was electrophoresed with a 1.0% agarose gel, DNA fragmentswith sizes from 3.7 to 9.0 kb were recovered from the gel, and endsthereof were bonded using T4 DNA ligase under a low DNA concentration(500 ng/2.0 ml) condition. Inverse PCR was performed on the obtainedDNA. The primer used herein is an end region of Tol1-tyr (the bases 162to 133 (SEQ ID NO: 20) of the base sequence (SEQ ID NO: 10) of GenBankAccession No. D42062). When a PCR product was electrophoresed, 10 ormore bands were generated per one reaction. The PCR product was insertedin a plasmid to form a clone and a base sequence thereof was examinedusing the same primer as the primer in the inverse PCR. Base sequencesaround insertion points of these genomic DNA clones are shown. Forreference, a sequence of a region corresponding to the donor plasmid isshown. 8 bp of TSD was observed in all of the insertion points.

FIG. 9 shows transposition frequencies of Tol1 and Tol2 in HeLa cells.Amounts of a donor and a helper were variously combined, and measurementof transposition frequencies was performed on Tol1 (left panel) and Tol2(right panel). A donor plasmid of Tol2 was prepared based on a donorplasmid of Tol1. Specifically, the Tol1 left arm in the Tol1 donorplasmid was replaced with the bases 1 to 755 of the Tol2 base sequence(GenBank Accession No. D84375,http://www.ncbi.nlm.nih.gov/Genbank/index.html, SEQ ID NO: 9) , and theright arm thereof was replaced with the bases 4147 to 4682 of the same.pHel03 in Cited document 33 was used for the helper plasmid of Tol2. Thewhole amount of the plasmid DNA was set to be 1,000 ng in eachmeasurement. Amounts of the donor plasmid and the helper plasmid areshown below the graphs. An insufficient amount from 1,000 ng wascomplemented with a filler plasmid (description of the filler plasmidamount is omitted). Average values (±standard error) of the numbers ofcolonies found from three independent measurements are shown in thegraphs.

FIG. 10 shows a test using HeLa cells regarding mutual effects betweenTol1 and Tol2. Plasmid DNA was combined and a measurement oftransposition frequencies was performed. Six kinds of combinationsdescribed below the graph were prepared. The whole amount of plasmid DNAwas set to 1,000 ng in each measurement. Description of the amount ofthe filler plasmid is omitted. It was found from the graph that a Tol1transposase and a Tol2 transposase transfer only corresponding elements,respectively.

FIG. 11 shows the base sequence (DDBJ/EMBL/GenBank Accession No.AB264112, SEQ ID NO: 3) of cDNA (whole length 2,900 bp) of a Tol1transposase and a deduced amino acid sequence (whole length 851aa, SEQID NO: 2).

FIG. 12 shows continuation of FIG. 11.

FIG. 13 shows continuation of FIG. 12.

FIG. 14 shows variation in a length of naturally occurring Tol1 element.PCR was performed in order to amplify an internal Tol1 portion per 130clones of genomic DNA that hybridize with both Tol1 end regions. Theprimers used were 30 by in the left end (1st to 30th bases) and 30 by ofthe right end (1855th to 1826th bases) of Tol1 and a piece of a bacteriacolony was used for a template. Conditions of the PCR were as follows:[94° C., 2 minutes], 30×[94° C., 20 seconds; 64° C., 20 seconds; 72° C.,2 minutes], and [72° C., 5 minutes]. The reaction solution after PCR waselectrophoresed with a 1% agarose gel, and lengths of the productsobtained from clones in which amplification occurred were recorded.Subsequently, PCR was performed on clones in which amplification did notoccur by prolonging a part of an elongation reaction (8 minutes insteadof 2 minutes in the previous time). Then, the reaction solution waselectrophoresed with a 0.8% agarose gel and the length of the productwas recorded. In the third PCR, an elongation reaction was carried outfor a longer period (20 minutes) and the reaction solution waselectrophoresed with 0.6% agarose gel. Lengths of totally 114 Tol1elements were thus revealed and their distributions were illustrated.

FIG. 15 shows a donor plasmid and a helper plasmid. FIG. 15 a shows aprocedure for producing a short donor plasmid. pDon1855 is a clonecontaining the whole region of Tol1-tyr element and 8 bp of target siteduplication (base sequence is CCTTTAGC), using pUC19 as a vector. Theclone was used as a template and PCR for forming a short donor wasperformed. A primer was designed so as to conform to the sequence of aTol1 end region, directed outward each other, and had a breaking site ofSalI in the 5′ end region. A DNA fragment of the product was cut withSalI, and the both ends were connected with the T4 DNA ligase and onceformed into a cyclic plasmid. A neomycin-resistant gene was inserted ina SalI site of the plasmid. The neomycin-resistant gene was obtained byamplifying a part of the plasmid pCMV-Tag1 (1675 to 3474 bases of DDBJfile AF025668) with a primer added with a Sail site. Black trianglesshown in the drawing indicate end inverted repeat sequences, whitetriangles indicate target site duplications, and gray triangles indicatePCR primers. FIG. 15 b shows helper plasmids. pHel1851aa was a completehelper. The helper was produced by inserting the whole region of thesequence encoding a Tol1 transfer enzyme (851 amino acids (SEQ ID NO:2), 31 to 2817 bases of DDBJ file AB264112) between the CMV promoter ofthe plasmid pCI (Promega Corp., Madison, Wis., USA) and a poly-Aadditional signal. pHel316aa is a defective helper. The helper wasproduced by adding modification to a part of the base sequence ofpHel851aa by PCR. The sequence in a part of the 996 to 1001 bases ofAB264112 is ATGAAA, which corresponds to amino acids, methionine andlysine, in a transfer enzyme. This part was changed to TAGTAA and twotermination codons were contained in the middle of the reading frame ofthe transfer enzyme.

FIG. 16 shows transposition frequencies of Tol1 elements having internaldeletion. Regarding the examined Tol1 element, only Tol1 arms areillustrated on the left of the drawing. However, the scale is notunified in the whole region. These were used as donors and inserted in acell together with the complete helper pHel851aa (hatched rectangle) andthe defective helper pHel316aa (while rectangle) to examine thetransposition frequency. Average colony numbers from three measurementsare shown in the graph. Horizontal lines in the graph are standarderrors of the average values.

FIG. 17 shows donor plasmids having Tol1 element with different lengths.pDon263Mcs shown in the bottom is a basic vector having a cloning site.All of restriction enzyme sites inherent to pUC19 were removed inadvance. However, only a HindIII site was not removed. Six kinds ofrestriction enzyme sites were provided in a bonding portion of the Tol1left and right arms as shown in the drawing. The above-describedmodifications were all made by preparing a 5′-end modified primer so asto achieve an object of each stage and performing PCR. pDon263McsNeoshown in the middle column was obtained by inserting aneomycin-resistant gene in a KpnI site and a PstI site of pDon263Mcs.Rectangles shown in the upper column indicate DNA fragments crammed toform long Tol1 elements. These fragments were produced by amplifyingvarious parts of bacteriophage λ (DDBJ file J02459) by PCR. Figuresshown blow the rectangles are numbers of nucleotides of the amplifiedportions. A PCR primer used for amplification was provided with an EcoRIor HindIII site in the 5′-end. The amplified product was cut with EcoRIor HindIII and then inserted in each site of pDon263McsNeo.

FIG. 18 shows transposition frequencies of long Tol1 element.Transposition frequencies were measured in the case of combination withthe complete helper pHel851aa (hatched rectangle) and the case ofcombination with the defective helper pHel316aa (white rectangle).Donors are shown in the ExHy mode below the rectangles. Average colonynumbers in three measurements are shown in the graph. Vertical linesindicate average standard errors.

FIG. 19 shows base sequences around insertion points. Genomic DNA wasextracted from a line derived from two colonies (N1 and N2) that hadbecome neomycin-resistant and assimilated with HindIII. Since Tol1element of a donor plasmid does not have a HindIII site in its inside,it is considered that Tol1 element will never be divided. Theassimilated DNA was electrophoresed with a 0.8% agarose gel, and DNAfragments of portions corresponding to 10 to 30 kb were recovered andends were then bonded with T4 DNA ligase in a low concentration state(100 ng/500 μl). Inverse PCR was performed using this DNA as a template.The used primers were portions corresponding to parts of respective armsof Tol1 (130 to 101st bases and 1758 to 1787th bases of D42062). Theconditions of PCR were as follows: [94° C., 2 minutes], 36×[94° C., 20seconds; 64° C., 20 seconds; 72° C., 5 minutes], and [72° C., 5minutes]. The PCR product was once cloned to a plasmid, and the basesequence was examined with the same primer as that used in the inversePCR. Base sequences near insertion points are shown. Base sequences ofcorresponding portions of the donor plasmid are aligned to be shown forreference. It is found that 8 bp of a target site duplication was formedin an insertion point of a host. The boxed portions are parts used asPCR primers in an analysis of Tol1 element carried out later (the detailwill be illustrated in FIG. 20).

FIG. 20 shows the analysis of incorporated Tol1 element. FIG. 20 a showsamplification of Tol1 element. DNAs used as templates for PCR arepDon263McsNeoE20 (pDon) and two transformant lines (N1 and N2). Theparts shown in boxes in FIG. 19 were used as primers (P0 represents asequence corresponding to plasmid pDon, P1 represents a sequencecorresponding to cell line N1, and P2 represents a sequencecorresponding to cell line N2). PCR was performed with the combinationdescribed above the lanes of electrophoresis. The conditions of PCR wereas follows: [94° C., 2 minutes], 30×[98° C., 10 seconds; 68° C., 20seconds], and [68° C., 10 minutes]. The electrophoresis was performedwith a 0.8% agarose gel and 2 μl out of 20 μl of the reaction solutionwas flowed. A DNA fragment as a PCR product was generated only in thecase of a right combination of a template and a primer. FIG. 20 b showscomparison of restriction enzyme break patterns. After precipitating thePCR product with ethanol, the PCR product was dissolved in distilledwater in an amount calculated so as to have approximately the same finalDNA concentration. The resultant PCR product was cut with restrictionenzymes BamHI and KpnI, and electrophoresed with a 1.0% agarose gel. Thewhole region of the base sequence of pDon263McsNeoE20 has been known andif it is cut with BamHI and KpnI, five fragments from 1.5 kb to 11.7 kbare generated. A cutting pattern observed in a PCR product from atransformant cell line was the same as in the case of the donor plasmid.

FIG. 21 shows structures of an indicator and a helper. plnd263GFP is anindicator and made of both ends of Tol1 element that was firstdiscovered (Tol1-tyr, 1855 bp, SEQ ID NO: 10) and a GFP gene insertedtherebetween. A plasmid holding them is pUC19. pHel851aa is a completehelper and prepared by inserting a sequence encoding a Tol1 transferenzyme (851 amino acids, SEQ ID NO: 2) between a CMV promoter of aplasmid pCI (Promega, Madison, Wis., USA) and poly-A additional signal.pHel316aa is a defective helper formed by changing apart of nucleotidesby PCR. The sequence of a portion from the 996th to 1001st bases isATGAAA, which corresponds to amino acids, methionine and lysine, in thetransfer enzyme. This sequence was changed to TAGTAA so that twotermination codons appeared in the middle of ORF of the transfer enzyme.In the drawing, brevity codes representing the GenBank file and numbersof nucleotides are added as explanation at each part constituting theplasmid. The brevity codes are as follows: [Tol1 ] D84375 (Tol1-tyrelement), [TPase] AB264112 (transfer enzyme gene), [pEGFP] U55763(plasmid pEGFP-C1; Clontech Laboratories, Mountain View, Calif., USA).TSD shows target site duplication, and the sequence thereof is CCTTTAGC.P_(CMV) represents a CMV promoter and PA represents a poly-A additionalsignal. The heavy line having a pointy right edge is a reading framecontained in the complete and defective helpers. The black triangleindicates an end inverted repeat sequence present in the Tol1-tyrelement. The small white triangle indicates a position and a directionof a PCR primer used in detection of excision.

FIG. 22 shows detection of excision generated in an embryo of X. laevisby PCR. A1 to A12 are 12 embryos from the set A used in the PCRanalysis. B1 to B12 are 12 embryos from the set B in the same manner.The upper column shows a result of electrophoresis of a reactionsolution after performing PCR that amplifies the whole region of Tol1element. The conditions of PCR were as follows: [94° C., 120 seconds],33×[94° C., 20 seconds; 64° C., 20 seconds; 68° C., 150 seconds], and[68° C., 60 seconds]. 5 μl out of 20 μl of the reaction solution wassubjected to electrophoresis. The agarose concentration was 1.0%. Bandswith 2.4 kb were observed in all samples of A and B. The lower columnshows a result of PCR that efficiently amplifies a product of excision.The conditions were the same as in the first PCR except that the periodof the elongation reaction part was shortened to 40 seconds. Inaddition, the agarose concentration in the electrophoresis was set to2.0%. Bands with a size close to 535 by were observed in samples A1 toA12. The same band was not observed in samples B1 to B12.

FIG. 23 shows base sequences around a breaking point of excision. plndin the uppermost column is the sequence of plnd263GFP shown forreference. TSD indicates target site duplication. This is contained inplnd263GFP from the first. Nucleotides enclosed within parentheses arenucleotides that are not present in plnd263GFP. There are also partsindicating only the length with [ ] since sequences are too long to beexpressed within the drawing. The sequences of corresponding portionsare as follows: [60 bp], the 504th to 445th bases of L09137 (pUC19), and[30 bp], the 1821st to 1850th bases of D84375 (Tol-tyr).

FIG. 24 illustrates a genealogical tree of the animal world. The drawingillustrates a standard genealogical tree based on phylum or subphylum asa unit.

FIG. 25 illustrates an overall flow of an experiment. The flow isdivided into two paths in mid-course. The left side illustratesdetection of excision, and the right side illustrates detection ofinsertion.

FIG. 26 illustrates plasmids used as templates for RNA synthesis.Plasmid pTem851aa was prepared by interleaving a sequence encoding aTol1 transfer enzyme (nt 31 to 2817 of DDBJ file AB031079) with pSP64Poly (A) Vector (Promega corp.). “Pro” indicates a SP6 promoter and “An”indicates a poly (A) sequence. When RNA synthesis is carried out usingpTem851aa as a template, RNA (mRNA851aa) made of about 2,900 nucleotidesis formed. This RNA encodes a full-length transfer enzyme. A portion ofsix bases (ATGAAA) in the middle corresponds to amino acids, methionineand lysine. Plasmid pTem316aa was obtained by changing the portion ofsix bases into two termination codons (TAGTAA). RNA (mRNA316aa) with thesame length is formed from this plasmid and the RNA encodes amino acidsright before the termination codons.

FIG. 27 shows a donor plasmid. White parts indicate a part of atyrosinase gene (DDBJ file AB010101) of medaka fish. The black partindicates Tol1 element (DDBJ file D84375). Triangles indicate positionsand directions of a primer for detecting excision. Base sequences are asfollows: Pex1: 3,594 to 3,623 of AB010101; Pex2: 3,866 to 3,895 ofAB010101; Pin1: 1,758 to 1,787 of D84375; and Pex2: 101 to 130 ofD84375.

FIG. 28 shows PCR to detect excision. PCR was performed using DNAsexpressed in respective lanes as templates. The DNA amounts of thetemplates were 10 pg for a donor plasmid, and a content corresponding toone embryo for DNA recovered from an embryo. Primers were Pex1 and Pex2.The upper column shows PCR to confirm that the donor plasmid wasrecovered. The conditions were set to [94° C. for 120 seconds], 25×[94°C. for 20 seconds; 64° C. for 20 seconds; 72° C. for 150 seconds], and[72° C. for 150 seconds]. The lower section is PCR to detect excision.The conditions were set to [94° C. for 120 seconds], 40×[94° C. for 20seconds; 64° C. for 20 seconds; 72° C. for 20 seconds], [72° C. for 20seconds].

FIG. 29 shows base sequences of PCR products in excision detection. The“donor” indicates a base sequence of both ends and portions subsequentthereto of Tol1 element on the donor plasmid. “Tol1” indicates Tol1element, “tyrosinase” indicates a portion derived from a tyrosinase geneof medaka fish, and “TSD” indicates target site duplication. “A1,” “A2”and “A3” indicate PCR products of respective treated sections. WholeTol1 element regions were deleted in all of the three samples, and othersequences were contained in the regions. Lengths thereof are describedin the drawing and respective base sequences are shown in the bottom.

FIG. 30 shows base sequences of clones detected as insertions. The“donor” indicates a base sequence of both ends and portions subsequentthereto of Tol1 element on the donor plasmid. “Clone 1” and “clone 2”indicate base sequences of corresponding portions of two clones obtainedby an inverse PCR technique. A part of Tol1 element is expressed byoutline characters on a black background.

BEST MODE FOR CARRYING OUT THE INVENTION

For the sake of simplicity of description, definitions and meanings ofsome terms used in the present specification will be collectivelydescribed in the following.

Inclusive expressions such as terms “contain” or “comprise” are used asexpressions also including meanings such as “consist” or “is/are.”

A “base sequence encoding an amino acid sequence” in the presentinvention refers to a base sequence capable of giving a protein havingthe amino acid sequence in the case of expressing a polynucleotide madeof the base sequence. Therefore, as long as the base sequence has asequence corresponding to an amino acid sequence, the base sequence mayhave a sequence portion that does not correspond to the amino acidsequence. Further, degeneracy of a codon is also naturally considered.In the expression an “amino acid sequence encoded by a base sequence,”degeneracy of a codon is also naturally taken into consideration.

The term “polynucleotide” refers to a polynucleotide in any form such asDNA and PNA (peptide nucleic acid), and RNA. The polynucleotide in thepresent invention is preferably DNA or mRNA.

The term “isolated” in the present invention is used interchangeablywith “purified.” “Isolated” when being used in terms of the transposaseof the present invention refers to, in the case that the transposase ofthe present invention is derived from natural materials, a statesubstantially free of components other than the enzyme among the naturalmaterials (substantially free of an impure protein in particular).Specifically, for example, in the isolated transposase of the presentinvention, the content of an impure protein is less than about 20% basedon the whole by weight conversion, preferably less than about 10%, morepreferably less than about 5%, and further more preferably less thanabout 1%. On the other hand, the term “isolated” in the case that thetransposase of the present invention is prepared by a geneticengineering technique refers to a state substantially free of othercomponents derived from the host cells used, a culture solution, and thelike. Specifically, for example, in isolated transposase of the presentinvention, the content of an impure component is less than about 20%based on the whole by weight conversion, preferably less than about 10%,more preferably less than about 5%, and furthermore preferably less thanabout 1%. In addition, a simple description of a “transposase” in thepresent invention means a “transposase in an isolated state” unless itis clear that a different meaning is expressed. The term “enzyme” usedin place of a transposase also means the same.

“Isolated” when being used for a polynucleotide typically refers to astate of being separated from other nucleic acids coexisting in anatural state when it is a naturally occurring polynucleotide. However,some other nucleic acid components such as a flanking sequence in anatural state (for example, a sequence of a promoter region, and aterminator sequence) may be contained. For example, in the state ofbeing “isolated” in the case of genomic DNA, other DNA componentscoexisting in a natural state are preferably substantially notcontained. On the other hand, in the state of being “isolated” in thecase of DNA prepared by a genetic engineering technique such as cDNAmolecules, cell components, culture solutions, and the like arepreferably substantially not contained. In the same manner, in the stateof being “isolated” in the case of DNA prepared by chemical synthesis,precursors (raw materials) such as dNTP, chemical substances used in asynthesis process, and the like are preferably substantially notcontained. In addition, a simple description of a “polynucleotide” inthe present specification means a polynucleotide in an isolated stateunless it is clear that a different meaning is expressed.

The term “DNA introduction” in the present specification means tointroduce DNA into a target cell no matter what is intended.Accordingly, genetic modifications (such as mutagenesis and genetargeting) are also included in the concept of DNA introduction.

(Tol1 Element Transposase)

A first aspect of the present invention provides a Tol1 elementtransposase based on achievement that a Tol1 element transposase wassuccessfully identified. A “Tol1 element transposase” refers to anenzyme capable of transferring Tol1 element that is a transposon foundin medaka fish. The term “transposase” when not particularly describedmeans the “Tol1 element transposase” hereinafter.

The transposase of the present invention in one embodiment has an aminoacid sequence encoded by the base sequence of SEQ ID NO: 1. As shown inexamples described later, the base sequence is a base sequence(including a termination codon) of ORF (open reading frame) encoding aTol1 element transposase. As a deduced amino acid sequence encoded bythe ORF, the amino acid sequence of SEQ ID NO: 2 (851 amino acids) wasobtained. Based on this fact, another embodiment of the presentinvention includes a protein having the amino acid sequence of SEQ IDNO: 2 (FIGS. 11 to 13). Note that a cDNA sequence corresponding to theamino acid sequence (including poly-A, FIGS. 11 to 13, SEQ ID NO: 3) isregistered in DDBJ/EMBL/GenBank as the Accession No. AB264112 (notpublished as of Dec. 13, 2006).

The transposase of the present invention has high specificity to a basesequence of substrate DNA, and does not have any substantial action toTol2 element that is a transposon found in medaka fish similarly to Tol1element.

In general, when a part of an amino acid sequence of a protein ismodified, the protein after modification may have equivalent functionsto those of the protein before modification. That is, modification of anamino acid sequence does not give a substantial effect on functions of aprotein, and the protein functions before modification may be kept aftermodification. Thus, another aspect of the present invention provides aprotein having an amino acid sequence homologous to the amino acidsequence of SEQ ID NO: 2 and having an enzymatic activity fortransferring Tol1 element (hereinafter also referred to as a “homologousprotein”). The “homologous amino acid sequence” herein refers to anamino acid sequence having partial difference from the amino acidsequence of SEQ ID NO: 2, in which the difference, however, does notgive any substantial influence on the protein functions (herein,enzymatic activity for transferring Tol1 element).

The “partial difference of an amino acid sequence” means occurrence ofvariation (change) in an amino acid sequence by deletion or substitutionof one to several amino acids constituting an amino acid sequence, oraddition or insertion of one to several amino acids, or combinationthereof. The difference of an amino acid sequence herein is acceptableas long as an enzymatic activity for transferring Tol1 element isretained (some fluctuation in activity is acceptable). As long as thiscondition is satisfied, a position at which an amino acid sequence isdifferent is not particularly limited, and the difference may begenerated in plural positions. “Plurality” herein is, for example, thenumber that corresponds to less than about 30% of the entire aminoacids, preferably the number that corresponds to less than about 20%,more preferably the number that corresponds to less than about 10%,further more preferably the number that corresponds to less than about5%, and most preferably the number that corresponds to less than about1%. That is, a homologous protein has an identity of, for example, about70% or more, preferably about 80% or more, more preferably about 90% ormore, further more preferably about 95% or more, and most preferablyabout 99% or more of the amino acid sequence of SEQ ID NO: 2.

It is preferable that a homologous protein is obtained by generatingpreservative amino acid substitution in an amino acid residue that isnot essential to an enzymatic activity for transferring Tol1 element.The “preservative amino acid substitution” herein refers to substitutionof an amino acid residue into an amino acid residue having a side chainwith similar nature. Amino acid residues are classified into severalfamilies by side chains thereof, such as basic side chains (e.g.,lysine, arginine, and histidine), acidic side chains (e.g., asparticacid and glutamic acid), uncharged polar side chains (e.g., glycine,asparagine, glutamine, serine, threonine, tyrosine, and cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, and tryptophan), β-branched sidechains (e.g., threonine, valine, and isoleucine), and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).Preservative amino acid substitution is preferably substitution betweenamino acid residues in the same family.

By the way, an identity (%) of two amino acid sequences or two basesequences (hereinafter, “two sequences” is used as the term includingthem) can be determined by the following procedure, for example. First,two sequences are aligned so that the sequences can be optimallycompared (for example, a gap may be introduced in the first sequence tooptimize alignment with the second sequence). When a molecule (aminoacid residue or nucleotide) at a specific position in the first sequenceis the same as a molecule at a position corresponding thereto in thesecond sequence, the molecules in these positions would be the same. Anidentity of two sequences is a function of the number of identicalpositions common in the two sequences (i.e. identity (%)=number ofidentical positions/total number of positions×100), and the number andsizes of gaps required for optimization of alignment should also bepreferably taken into consideration.

Comparison of two sequences and determination of identity thereof arefeasible using a mathematical algorithm. Specific examples of themathematical algorithm applicable to comparison of sequences include analgorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci.USA 87: 2264-68 and modified in Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-77, but are not limited thereto. Such analgorithm is incorporated into the NBLAST program and XBLAST program(version 2.0) described in Altschul et al. (1990) J. Mol. Biol.215:403-10. In order to obtain an amino acid sequence homologous to aspecific amino acid sequence, for example, BLAST polypeptide search maybe carried out by the XBLAST program at a score of 50 and a wordlengthof 3. In order to obtain a base sequence homologous to a specific basesequence, for example, BLAST nucleotide search may be carried out by theNBLAST program at a score of 100 and a wordlength of 12. In order toobtain a gap alignment for comparison, Gapped BLAST described inAltschul et al. (1997) Amino Acids Research 25(17): 3389-3402 isavailable. When BLAST and Gapped BLAST are employed, a default parameterof a corresponding program (such as XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov for detail. Examples of another mathematicalalgorithm applicable to comparison of sequences include the algorithmdescribed in Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Suchan algorithm is incorporated in the ALIGN program available from, forexample, the GENESTREAM network server (IGH Montpellier, France) or theISREC server. When the ALIGN program is used for comparison of aminoacid sequences, for example, the PAM120 residue mass table is used, agap length penalty can be set to 12 and a gap penalty can be set to 4.

An identity of two amino acid sequences can be determined with the GAPprogram of the GCG software package using Blossom 62 matrix or PAM250matrix, and setting a gap load to 12, 10, 8, 6 or 4 and a gap lengthload to 2, 3 or 4. Further, a homology of two nucleic acid sequences canbe determined with the GAP program of the GCG software package(available from http://www.gcg.com) setting a gap load to 50 and a gaplength load to 3.

The transposase of the present invention can be easily prepared by agenetic engineering technique. For example, a suitable host cell (suchas Bacillus coli) is transformed with a polynucleotide encoding thetransposase of the present invention and a protein expressed in thetransformant is recovered to thereby prepare the transposase. Therecovered protein is appropriately purified in accordance with theapplications. Thus, if the transposase of the present invention is to beobtained as a recombinant protein, various modifications are possible.For example, DNA encoding the transposase of the present invention andanother suitable DNA are inserted in the same vector and a recombinantprotein is produced using the vector, which enables to obtain thetransposase made of a recombinant protein connected to any peptide orprotein. Further, addition of a sugar chain and/or lipid, ormodification to generate processing of N ends or C ends may be provided.According to modifications as described above, extraction of arecombinant protein, simplification of purification, addition ofbiological functions, and the like are possible.

Note that a method for preparing the transposase of the presentinvention is not limited to genetic engineering techniques. For example,if materials exist in nature, the transposase of the present inventioncan also be prepared from the natural materials by a standard technique(fracture, extraction, purification, etc.). In addition, the transposaseof the present invention is generally prepared in an isolated state.

(Polynucleotide Encoding Tol1 Element Transposase)

A second aspect of the present invention provides a polynucleotideencoding the transposase of the invention. In one embodiment, thepolynucleotide of the present invention is made of a base sequenceencoding the amino acid sequence of SEQ ID NO: 2. Specific examples ofthe base sequences are shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ IDNO: 4. The base sequence of SEQ ID NO: 1 is a sequence found as ORFencoding a Tol1 element transposase. Further, the base sequence of SEQID NO: 3 corresponds to a sequence found as full-length cDNA encoding aTol1 element transposase. The base sequence of SEQ ID NO: 4 correspondsto a genomic DNA sequence (4355 base pairs, a target site duplicatedsequence (TSD) is not included) for the full-length cDNA.

Herein, in general, in the case that a part of a polynucleotide encodinga protein is subjected to modification, the protein encoded by thepolynucleotide after modification may have equivalent functions to theprotein encoded by the polynucleotide before modification. That is,modification of a base sequence may not give a substantial effect onfunctions of the encoded protein, and the encoded protein functions maybe kept before and after the modification. Thus, another embodiment ofthe present invention provides a polynucleotide made of a base sequencehomologous to the base sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, orSEQ ID NO: 4, and encoding a protein having an enzymatic activity fortransferring Tol1 element (hereinafter also referred to as a “homologouspolynucleotide”). The “homologous base sequence” herein refers to a basesequence having a partial difference from the base sequence of any ofSEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4, in which the difference,however, gives no substantial effect on a function of the proteinencoded by the base sequence (the function means an enzymatic activityfor transferring Tol1 element herein).

Specific examples of a homologous polynucleotide include apolynucleotide that hybridizes to a polynucleotide made of a basesequence complementary to the base sequences of any of SEQ ID NO: 1, SEQID NO: 3, and SEQ ID NO: 4 under stringent conditions. The “stringentconditions” herein refers to conditions where a so-called specifichybrid is formed, but a non-specific hybrid is not formed. Suchstringent conditions have been known to a skilled person, and can be setin reference to, for example, Molecular Cloning (Third Edition, ColdSpring Harbor Laboratory Press, New York) and Current protocols inmolecular biology (edited by Frederick M. Ausubel et al., 1987). Thestringent conditions include, for example, such conditions thatincubation is carried out at about 42° C. to about 50° C. using ahybridization solution (50% formamide, 10×SSC (0.15 M NaCl, 15 mM sodiumcitrate, pH 7.0), 5×Denhardt solution, 1% SDS, 10% dextran sulfate, 10μg/ml of modified salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)),and then washing is carried out at about 65° C. to about 70° C. with0.1×SSC and 0.1% SDS. Preferable stringent conditions include, forexample, such conditions that 50% formamide, 5×SSC (0.15 M NaCl, 15 mMsodium citrate, pH 7.0), 1×Denhardt solution, 1% SDS, 10% dextransulfate, 10 μg/ml of modified salmon sperm DNA, 50 mM phosphate buffer(pH 7.5) is used as a hybridization solution.

Other specific examples of a homologous polynucleotide include apolynucleotide made of a base sequence containing substitution,deletion, insertion, addition, or inversion of one or plural bases basedon the base sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ IDNO: 4, and encoding a protein having an enzymatic activity fortransferring Tol1 element. Substitution and deletion of bases may occurin a plurality of sites. “Plurality” herein indicates, for example, 2 to40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 basesalthough depending on a position and a kind of an amino acid residue ina conformation of a protein encoded by the polynucleotide. Such ahomologous polynucleotide as described above can be obtained bymodifying a polynucleotide having the base sequence of any of SEQ ID NO:1, SEQ ID NO: 3, and SEQ ID NO: 4 so as to contain substitution,deletion, insertion, addition and/or inversion of bases, utilizingintroduction of variation, for example, a restriction enzyme treatment,a treatment with exonuclease, DNA ligase, or the like, a site-specificmutation introduction method (Molecular Cloning, Third Edition, Chapter13, Cold Spring Harbor Laboratory Press, New York), and a randommutation introduction method (Molecular Cloning, Third Edition, Chapter13, Cold Spring Harbor Laboratory Press, New York). A homologouspolynucleotide can also be obtained by other methods such as exposure toultraviolet radiation.

Other examples of a homologous polynucleotide include a polynucleotidein which such difference in bases as described above is recognized dueto a polymorphism typified by SNP (monobasic polymorphism).

The polynucleotide of the present invention can be prepared to be in anisolated state by using a standard genetic engineering technique,molecular biological technique, biochemical technique, or the like, inreference to sequence information disclosed in the present specificationor attached sequence listings. Specifically, the polynucleotide of thepresent invention can be prepared from a medaka fish (Oryzias laptipes)genomic DNA library or cDNA library, or a cell extract of medaka fish,by appropriately using an oligo-nucleotide probe/primer capable ofspecifically hybridizing to the polynucleotide of the present invention.The oligo-nucleotide probe/primer can be easily synthesized using acommercially available automated DNA synthesis device. For a productionmethod of a library used for preparing the polynucleotide of the presentinvention, for example, Molecular Cloning, Third edition, Cold SpringHarbor Laboratory Press, New York can be referred to.

For example, a polynucleotide having the base sequence of SEQ ID NO: 3can be isolated from a medaka fish cDNA library utilizing ahybridization method in which whole or a part of the base sequence or asequence complementary thereto is used as a probe. Also, thepolynucleotide can be amplified and isolated utilizing a nucleic acidamplification reaction (for example, PCR) using a synthesizedoligo-nucleotide primer designed so as to specifically hybridize to apart of the base sequence.

(Expression Construct Containing Tol1 Element Transposase)

A further aspect of the present invention relates to an expressionconstruct containing the polynucleotide of the present invention. Apromoter is preferably incorporated in the expression construct of thepresent invention. However, when a polynucleotide contained in theexpression construct essentially has a promoter region, a promoter canbe omitted.

The promoter is operably linked to the polynucleotide of the presentinvention. In the expression construct with such a constitution, anaction of the promoter enables the polynucleotide of the presentinvention to be forcibly expressed in a target cell. Herein, “a promoteris operably linked to a specific polynucleotide sequence” has the samemeaning as that of “a specific polynucleotide sequence is arranged undercontrol of a promoter,” and generally, a specific polynucleotidesequence is linked to the 3′-end side of the promoter directly or viaanother sequence.

For the promoter, CMV-IE (cytomegalovirus initial gene-derivedpromoter), SV40ori, retrovirus LTP, SRα, EF1α, β actin promoters, andthe like can be used. Mammal tissue specific promoters such as anacetylcholine receptor promoter, an enolase promoter, an L7 promoter, anestin promoter, an albumin promoter, an alpha-fetoprotein promoter, akeratin promoter, and an insulin promoter may be used.

In the expression construct of the present invention, a poly-Aadditional signal sequence, a poly-A sequence, an enhancer sequence, aselective marker sequence, and the like can also be arranged. Stabilityof mRNA generated from the expression construct is improved by use of apoly-A additional signal sequence or a poly-A sequence. The poly-Aadditional signal sequence or the poly-A sequence is connected to thepolynucleotide of the present invention in the downstream side. On theother hand, improvement of an expression efficiency is intended with useof an enhancer sequence. Further, when an expression constructcontaining a selective marker sequence is used, presence or absence (anda degree thereof) of introduction of the expression construct can beconfirmed utilizing a selective marker.

Note that insertion operations, and the like of a promoter, thepolynucleotide sequence of the present invention, an enhancer sequence(if necessary), and a selective marker sequence (if necessary) can beperformed by a standard recombinant DNA technique such as a method usinga restriction enzyme and a DNA ligase (e.g., Molecular Cloning, ThirdEdition, 1.84, Cold Spring Harbor Laboratory Press, New York can bereferred to).

The expression construct of the present invention can be used tointroduce the polynucleotide sequence of the present invention into atarget cell. Although the form of the expression construct is notparticularly limited as long as it can be used for such a purpose, theexpression construct preferably takes a form of an expression vector.The “expression vector” herein refers to a nucleic acid molecule capableof introducing a polynucleotide inserted therein into a desired cell(target cell) and expressing the polynucleotide in the cell, andincludes a virus vector and a nonvirus vector. A gene introductionmethod using a virus vector skillfully utilizes a phenomenon ofinfection of a cell with a virus, and a high gene introductionefficiency can be obtained. As the virus vector, an adenovirus vector,an adeno-associated virus vector, a retrovirus vector, a lentivirusvector, a herpesvirus vector, a Sendai virus vector, and the like, havebeen developed. In an adeno-associated virus vector, a retrovirusvector, and a lentivirus vector among the above virus vectors, foreigngenes incorporated into a vector are incorporated into a hostchromosome, and stable and long-term expression can be expected. Sincethe case of a retrovirus vector requires cell division for incorporationof a virus genome into a host chromosome, a retrovirus vector is notappropriate for gene introduction into a nondividing cell. On the otherhand, a lentivirus vector and an adeno-associated virus vector causeincorporation of foreign genes into host chromosomes after infectionalso in nondividing cells. Therefore, these vectors are effective forexpressing foreign genes stably and for a longtime in nondividing cellssuch as nerve cells and liver cells.

Each virus vector can be prepared by following a reported method orusing a commercially available special kit. For example, preparation ofan adenovirus vector can be carried out by a COS-TPC method or afull-length DNA introduction method. The COS-TPC method is a method forpreparing a recombinant adenovirus by transfecting a recombinant cosmidin which a desired cDNA or an expression cassette are incorporated and aparent virus DNA-terminal protein complex (DNA-TPC) to 293 cells at thesame time and utilizing homologous recombination generated in the 293cells (Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Takamori, K.,Tokuda, C., and Saito, I. (1996) Proc. Natl. Acad. Sci. USA, 93, 1320.).On the other hand, the full-length DNA introduction method is a methodfor producing a recombinant adenovirus by performing a restrictiondigestion treatment on a recombinant cosmid inserted with a desiredgene, thereafter transfecting the product to 293 cells (Miho Terashima,Saki Kondo, Yumi Kanegae, and Izumi Saito (2003) Experimental Medicine21 (7) 931). The COS-TPC method can be performed with the AdenovirusExpression Vector Kit (Dual Version) (TAKARA BIO INC.) and Adenovirusgenome DNA-TPC (TAKARA BIO INC.). Further, the full-length DNAintroduction method can be performed with Adenovirus Expression VectorKit (Dual Version) (TAKARA BIO INC.).

On the other hand, a retrovirus vector can be prepared by the followingprocedure. First, virus genomes (gag, pol, and env genes) other thanpackaging signal sequences between LTR (Long Terminal Repeat) present onthe both ends of a virus genome are removed, and a desired gene isinserted therein. The thus constructed virus DNA is introduced in apackaging cell constitutionally expressing gag, pol, and env genes. Withthis introduction, only a vector RNA having a packaging signal sequenceis incorporated in a virus particle and a retrovirus vector is produced.

As a vector obtained by application or improvement in an adeno vector, avector in which specificity is improved by modification of a fiberprotein (specific infection vector) and a gutted vector from whichimprovement in an expression efficiency of a desired gene can beexpected (helper-dependent vector), and the like, have been developed.The expression vector of the present invention can be constructed assuch a virus vector.

As a nonvirus vector, a liposome, a positively charged liposome(Felgner, P. L., Gadek, T. R., Holm, M. et al., Proc. Natl. Acad. Sci.,84:7413-7417, 1987), HVJ (Hamagglutinating virus of Japan)-liposome(Dzau, V. J., Mann, M., Morishita, R. et al., Proc. Natl. Acad. Sci.,93:11421-11425, 1996, Kaneda, Y., Saeki, Y. & Morishita, R., MolecularMed. Today, 5:298-303, 1999), and the like have been developed. Theexpression vector of the present invention can be constructed as such anonvirus vector.

(DNA Introduction System Utilizing Tol1 Element)

Another aspect of the present invention relates to a DNA introductionsystem utilizing Tol1 element. The DNA introduction system of thepresent invention can be used for introducing specific DNA into a targetcell. In other words, specific DNA can be introduced into genomic DNA ofa target cell using the DNA introduction system of the presentinvention. The DNA introduction system of the present invention is thusutilized as a means for genetic manipulation such as gene introductionand gene modification.

The DNA introduction system of the present invention includes a donorfactor and a helper factor. The donor factor and the helper factorpreferably exist in the system as distinct constituent factors. That is,it is preferable that the donor factor and the helper factor are in aphysically separated state. However, the donor factor and the helperfactor may coexist in a single structural body.

The donor factor delivers a desired DNA to a target cell, and has astructure in which the desired DNA is inserted in transposasegene-defected Tol1 element.

The “target cell” means a cell to which the DNA introduction system ofthe present invention is applied, that is, a cell to be an object ofgenetic manipulation using the DNA introduction system of the presentinvention. The “target cell” herein indicates a vertebrate cell andspecific examples thereof include various cells of mammals (e.g., human,monkey, cattle, horse, rabbit, mouse, rat, guinea pig, and hamster),birds (e.g., chicken and quail), fish (e.g., medaka and zebrafish), andamphibians (e.g., frog). As the target cell, the following examples canbe used: a myocardial cell, a smooth muscle cell, an adipose cell, afibrocyte, a bone cell, a chondrocyte, an osteoclast, a parenchymalcell, an epidermal keratinocyte (keratinocyte), epithelial cells (e.g.,skin epidermal cell, corneal epithelial cell, conjunctival epithelialcell, oral mucosal epithelium, follicle epithelial cell, oral mucosalepithelial cell, airway mucosal epithelial cell, and intestinal mucosalepithelial cell), endothelial cells (e.g., corneal endothelial cell andvascular endothelial cell), a nerve cell, a glial cell, a splenic cell,a pancreatic β cell, a mesangium cell, a Langerhans cell, a liver cell,or precursor cells thereof, or mesenchymal stem cells (MSC), embryonicstem cells (ES cells), embryonic germ cells (EG cells), adult stemcells, fertilized eggs, and the like. In addition to normal cells, thefollowing cells can be used as target cells: cells that develop someabnormalities such as cancer cells, or established cell lines such asHeLa cells, CHO cells, Vero cells, HEK293 cells, HepG2 cells, COS-7cells, NIH3T3 cells, Sf9 cells, and the like.

The DNA introduction system of the present invention is applied to atarget cell in an isolated state or a target cell in a state of aconstituent factor of a biological organism. Therefore, the presentinvention can be carried out under any of in vitro, in vivo, and ex vivoenvironments. “Isolated” used herein refers to a state of being takenout from the original environment thereof (for example, a state ofconstituting a biological organism). Accordingly, in general, anisolated target cell exists in a culture container or a preservationcontainer, and the cell can be artificially manipulated in vitro.Specifically, a cell being separated from a living body and cultured exvivo (including an established cell line) has eligibility as an isolatedtarget cell. In addition, as long as a target cell is in the isolatedstate in the above-described meaning, the target cell is an isolatedcell even in a state of a constituent factor of a tissue body.

The “isolated target cell” can be prepared from a biological organism.On the other hand, cells obtained from RIKEN BioResource Center(independent administrative institution), the National Institute ofTechnology and Evaluation Institute (independent administrativeinstitution), ATCC (American Type Culture Collection), DSMZ (GermanCollection of Microorganisms and Cell Cultures), and the like can beused as isolated target cells.

In one embodiment of the present invention, the DNA introduction systemof the invention is applied to vertebrate cells other than cells in astate of a constituent factor of a human individual. That is, in thisembodiment, when the DNA introduction system is carried out, a cellbeing isolated from a human or a cell from vertebrates other than human(without regard to whether the cell is in a state of a constituentfactor of an organism or not) is a target cell.

Tol1 element used in the donor factor is a transposase gene-defectedTol1 element. “transposase gene-defected” means containing no functionaltransposase gene, and also includes a state where there are someremaining transposase genes as long as the transposase genes do notexert genetic functions, not limited to a state where the transposasegenes are completely deleted. That is, as a result of a change of a partof the sequence, transposase genes are in a state where the sequenceexcluding the portion associated with the change is left althoughfunctions thereof are lost, and such a state can also be included in themeaning of “transposase gene-defected.”

Tol1 element is a DNA-type element existing about 100 to 200 copies in agenome of medaka fish (Koga A., Sakaizumi M., and Hori H. (2002) ZoologSci 19: 1-6. (Cited document 10)), and discovered as a fragment insertedin a tyrosinase gene (Koga A., Inagaki H., Bessho Y., and Hori H. (1995)Mol Gen Genet 249: 400-405. (Cited document 11)). The sequence (SEQ IDNO: 10) of this fragment (Tol1-tyr, 1855 base pairs, not containing atarget site duplicated sequence (TSD)) is registered in GenBank as theAccession No. D42062. The inverted repeat sequence characteristic toTol1 element was identified from an analysis on Tol1-tyr (Koga A.,Sakaizumi M., and Hori H. (2002) Zoolog Sci 19: 1-6. (Cited document10)). In view of this finding, one preferable embodiment of the presentinvention uses Tol1 element having the inverted repeat sequence of SEQID NO: 5 in the 5′ end region and the inverted repeat sequence of SEQ IDNO: 6 in the 3′ end region. That is, in Tol1 element in this embodiment,the sequence made of 5′-cagtagcggttcta-3′ (SEQ ID NO: 5) is present inthe 5′ end region of its sense strand and, in the same manner, thesequence made of 5′-tagaaccgccactg-3′ (SEQ ID NO: 6) is present in the3′ end region of its sense strand. In addition, all Tol1 elementsreported so far including Tol1-tyr have defected transposase genes, andhave eligibility as Tol1 element in the present invention. As specificexamples of Tol1 element that can be used in the present invention, basesequences thereof are shown in SEQ ID NOs: 10 to 12. Note that the basesequence of SEQ ID NO: 11 is a clone sequence (969 base pairs) obtainedby removing internal 886 base pairs from Tol1-tyr and was confirmed tobe transposable similarly to Tol1-tyr. Further, the base sequence of SEQID NO: 12 is a clone sequence (297 base pairs) obtained by removinginternal 1576 base pairs from Tol1-tyr and adding recognition sequencesof six kinds of restriction enzymes in order to insert other DNAfragments and was confirmed to be transposable similarly to Tol1-tyr.

A modified element of any of these examples can also be used. The“modified element” herein indicates a polynucleotide molecule made of abase sequence homologous to the base sequence of any of SEQ ID NOs: 10to 12, and functions as a transposon similarly to the polynucleotidemolecule before modification. A transposase having the amino acidsequence of SEQ ID NO: 2 can bond to an end of the modified element.Regarding the term “homologous,” description in the section of the“polynucleotide encoding a Tol1 element transposase” can be referred to.

As shown in examples described later, it was revealed according tofurther studies made by the present inventors that a transpositionefficiency was not damaged also in the case of deleting internal 1592base pairs (from 158th base to 1749th base counting from the 5′-end)from Tol1-tyr. Based on this finding, one embodiment of the presentinvention uses Tol1 element made of 5′-end side DNA and 3′-end side DNA,which is obtained by deleting at least bases from 158th base to 1749thbase (1592 base pairs) counting from the 5′-end in the base sequence ofTol1-tyr (the base sequence of SEQ ID NO: 10). In other words, theembodiment uses a donor factor having a structure in which desired DNAis inserted between DNA of the 5′ end region of the base sequence of SEQID NO: 10 (157 base pairs at maximum length) and DNA of the 3′ endregion of the base sequence of SEQ ID NO: 10 (106 base pairs at maximumlength). By deleting an internal region unnecessary for transpositionability as much as possible, maximization of a loadable DNA size isintended. It was suggested that, when 1592 base pairs in the internalregion are deleted as described above, introduction of DNA with a sizeexceeding 20 kb is possible (examples described later). Herein, aspecific example of the 5′-end side DNA is DNA having the base sequenceof SEQ ID NO: 21 (157 base pairs), and a specific example of the 3′-endside DNA is DNA having the base sequence of SEQ ID NO: 22 (106 basepairs).

Tol1 element can be easily prepared by PCR, and the like, with a primerspecific to Tol1 element (see examples described later) using medakagenomic DNA as a template. Regarding details of preparation methods,refer to examples described later, or Koga A., Sakaizumi M., and Hori H.(2002) Zoolog Sci 19: 1-6, Tsutsumi M., Imai S., Kyono-Hamaguchi Y.,Hamaguchi S., Koga A., and Hori H. (2006) Pigment Cell Res 19: 243-247,and the like.

“A desired DNA” contained in the donor factor refers to DNA introducedin genomic DNA of a target cell by the DNA introduction system of thepresent invention. The DNA introduction system of the present inventioncan be utilized as a tool for gene introduction for the purposes offunctional analysis of genes, improvement and restoration (treatment) ofspecific functions, addition of new traits, differentiation induction,production of useful proteins (such as interferon, insulin,erythropoietin, and antibodies), formation of transgenic animals, andthe like. When the DNA introduction system of the present invention isused as the tool, specific genes are used as “desired DNA.” Examples ofthe genes herein can include genetic disease associated genes such as anadenosine deaminase (ADA) gene, a factor IX gene, agranulocyte-macrophage colony-stimulating factor (GM-CSF) gene, a p53cancer suppressing gene, a simple herpes virus thymidine kinase (HSV-tk)gene, a vascular endothelial growth factor (VEGF) gene, and ahepatocellular growth factor (HGF), genes encoding hormones such asinsulin and erythropoietin, genes encoding growth factors such asinterferon, an insulin-like growth factor, an epidermal growth factor(EGF), a fibrocyte growth factor (FGF), and interleukins, genes encodingantibodies (for therapy, diagnosis, detection, etc.), marker genes suchas a green fluorescent protein (GFP) gene, a β-galactosidase (lacZ)gene, a chloramphenicol-resistant (CAT) gene, and a luciferase (LUC)gene, and genes having unknown functions. In addition to genes existingin nature, genes obtained through artificial manipulation (artificialgenes) can also be used. Further, genes in use may be of the same typeas or different type from a target cell. Desired DNA encoding two ormore types of genes may be employed.

When genetic modification is intended, any DNA capable of destroying orinactivating a target cell, for example, a modified gene of the targetgene, is used as the “desired DNA.”

An insertion position of the “desired DNA” is not particularly limitedas long as no adverse effects are given to the function of a transposonof Tol1 element (transposition function). That is, the “desired DNA” maybe inserted in a position other than the both ends that are acting sitesof a transposase. Specifically, for example, endogenous restrictionenzyme recognition sites (e.g., SalI) existing in the region other thanboth ends may be utilized as an insertion site in Tol1 element.Alternatively, an insertion site may be artificially formed withoutusing an endogenous restriction enzyme recognition site.

In one preferable embodiment of the present invention, a target siteduplicated sequence is connected to the 5′-end and the 3′-end of Tol1element. The “target site duplicated sequence,” that is, TSD (targetsite duplication), indicates a tandem repeat sequence formed intransposition. When a transposon is inserted, since double stranded DNAis broken at different positions, the sequence between them isduplicated, which results in formation of TSD. In the case of Tol1element, TDS with 8 bp in one side is formed. For example, TSD havingthe sequence of any of SEQ ID NOs: 13 to 15 can be used in the presentinvention. In addition, the sequences of SEQ ID NOs: 13, 14 and 15correspond to TSD of Tol1-tyr, TSD of Tol1-L1, and TSD of Tol1-L2,respectively.

In order to achieve a high introduction efficiency, a vector in whichdesired DNA, Tol1 element, etc., are incorporated therein as anexpression cassette is preferably used as a donor factor. The kind ofthe vector used herein is not particularly limited. Regarding kinds,production methods, and the like of the vector, the above description(section of the expression construct of the present invention) can bereferred to.

The helper factor delivers a transposase into a target cell, andincludes the transposase of the invention (i.e., Tol1 elementtransposase) or the polynucleotide of the invention (i.e.,polynucleotide encoding the Tol1 element transposase). When the DNAintroduction system of the present invention is introduced into thetarget cell, a transposase supplied by the helper factor acts on Tol1element supplied by the donor factor. As a result, desired DNA insertedin the Tol1 element is incorporated in the genomic DNA of the targetcell.

In order to achieve a high introduction efficiency, the helper factoralso is preferably constructed as a vector similarly to the donorfactor. That is, it is preferable to use a vector in which an expressioncassette containing a polynucleotide encoding a Tol1 element transposaseis incorporated as the helper factor.

Introduction of the donor factor and the helper factor into a targetcell can be carried out by a calcium phosphate coprecipitation method,lipofection (Feigner, P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 84,7413-7417 (1984)), an HVJ liposome method, a DEAE dextran method,electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. U.S.A. 81,7161-7165 (1984)), microinjection (Graessmann, M. & Graessmann, A.,Proc. Natl. Acad. Sci. U.S.A. 73, 366-370 (1976)), a gene gun method, anultrasonic gene introduction method, etc., in consideration of kinds ofthe target cell, forms of the donor factor and the helper factor, andthe like. When a virus vector is used as an expression construct,introduction into the target cell is performed by infection.

The donor factor and the helper factor are not necessarily introducedinto a target cell simultaneously. However, it could be preferable thatthe both factors are simultaneously co-introduced into the target cellfrom the viewpoints of operationality and mutual actions of the bothfactors.

The present invention further provides uses of the DNA introductionsystem of the invention. One of the uses is a DNA introduction method.In the DNA introduction method of the present invention, a step ofintroducing the DNA introduction system of the invention is carried outon a target cell obtained from a vertebrate cell. Further, based on thefinding that Tol1 element and Tol2 element do not have an influence oneach other's transposition, provided is a DNA introduction methodcharacterized by further including the following step, that is, a stepof introducing DNA different from desired DNA, which is introduced usingTol1 element, into the target cell using Tol2 element. A DNAintroduction method utilizing Tol2 element can be carried out inreference to Koga A., Hori H., and Sakaizumi M. (2002) Mar Biotechnol 4:6-11. (Cited document 13), Johnson Hamlet M. R., Yergeau D. A., KuliyevE., Takeda M., Taira M., Kawakami K., and Mead P. E. (2006) Genesis 44:438-445. (Cited document 14), Choo B. G., Kondrichin I., Parinov S.,Emelyanov A., Go W., Toh W. C., and Korzh V. (2006) BMC Dev Biol 6: 5(Cited document 15), and the like. In addition, Cited document 13reports gene introduction using Tol2 element, Cited document 14 reportsmutagenesis using Tol2, and Cited document 15 reports trap of genes andpromoters or enhancers using Tol2 element, respectively.

The DNA introduction system of the present invention can be used intransgenic fish, transgenic mice, knockout mice, etc., for the purposeof producing genetically modified animals. For example, the DNAintroduction system of the present invention can be introduced into acytoplasm, a vitellus or a nucleus of a fertilized egg of zebra fish,medaka fish, etc., by a method such as microinjection to generatetransgenic fish.

On the other hand, by using the DNA introduction system of the presentinvention, a specific gene-introduced fertilized oocyte or embryo-stemcell can be produced, and a transgenic non-human mammal can be generatedfrom such a cell. The transgenic non-human mammal can be produced with amicroinjection method of directly injecting DNA into a pronucleus of afertilized egg, a method using a retrovirus vector, a method using an EScell, or the like. In the following, a method that utilizes amicroinjection method will be described as one example of a productionmethod of a transgenic non-human mammal.

In the microinjection method, a fertilized egg is first collected froman oviduct of a female mouse whose copulation was confirmed and thencultured, thereafter the DNA introduction system of the presentinvention is introduced into the pronucleus. The fertilized egg aftercompletion of the introduction operation is transplanted into an oviductof a pseudopregnant mouse, and the mouse after transplantation is bredfor a certain period of time to obtain a neonatal mouse (F0). In orderto confirm that the introduced genes are appropriately incorporated intochromosomes of the neonatal mouse, DNA is extracted from the tale or thelike of the neonatal mouse and the DNA is subjected to a PCR methodusing a primer specific to the introduced genes, a dot hybridizationmethod using a probe specific to the introduced genes, or the like.Although species of “the transgenic non-human mammal” in the presentspecification are not particularly limited, rodents such as mice andrats are preferable.

(Method of Transferring Specific DNA Site on Genomic DNA)

Another aspect of the present invention provides a method oftransferring a specific DNA site on genomic DNA of a target cell byusing the transposase of the present invention or a polynucleotideencoding the transposase. In one embodiment of this aspect, thetransposase of the present invention or the polynucleotide of thepresent invention is introduced into a cell (target cell) having Tol1element in which a polynucleotide encoding a transposase is deleted ongenomic DNA. The introduced transposase (or a transposase expressed fromthe introduced polynucleotide) acts on Tol1 element contained in thetarget cell to allow transposition. Not limited to a cell that has cometo have Tol1 element by artificial manipulation, a cell essentially(i.e. as an intrinsic element) having Tol1 element can be used as the“target cell” herein. That is, a cell to which the method of the presentinvention is applicable is not limited to a cell after undergoing anintroduction operation of Tol1 element.

When the method of the invention is applied to Tol1 element in whichanother polynucleotide sequence is inserted, influences and effects dueto transfer of the polynucleotide sequence can be examined, andbeneficial information about functions of the polynucleotide sequencecan be obtained. Thus, the method of the present invention is useful foran analysis of functions of various polynucleotides typically includinggenes. On the other hand, when the method of the present invention isapplied to Tol1 element in which no other polynucleotide sequence isinserted, functions of the Tol1 element itself and influences due toinsertion of the polynucleotide sequence, etc., can be examined. Thus,the method of the present invention is also useful in studies of Tol1element.

A transposase corresponding to either of Tol1 element and Tol2 elementenables selective transposition of one of two types of DNAs in cellsintroduced with the two types of DNAs by using Tol1 element and Tol2element. That is, it becomes possible to control two types of introducedDNAs independently. The present invention thus provides a method oftransferring a specific DNA site on genomic DNA, which includes a stepof providing a transposase corresponding to Tol1 element or Tol2 elementto a genetically manipulated cell with a DNA introduction methodutilizing Tol1 element and Tol2 element. The transposase may be forciblyexpressed in a target cell by introducing a polynucleotide encoding thetransposase into the target cell. Regarding introduction of a Tol2element transposase, Koga A., Hori H., and Sakaizumi M. (2002) MarBiotechnol 4: 6-11. (Cited document 13) , Johnson Hamlet M. R., YergeauD. A., Kuliyev E., Takeda M., Taira M., Kawakami K., and Mead P. E.(2006) Genesis 44: 438-445. (Cited document 14) , Choo B. G., KondrichinI., Parinov S., Emelyanov A., Go W., Toh W. C., and Korzh V. (2006) BMCDev Biol 6: 5 (Cited document 15) , and the like can be referred to. Theamino acid sequence of the Tol2 element transposase is of SEQ ID NO: 7.The cDNA sequence (not containing poly A) encoding the transposase andthe genomic DNA sequence (not containing TSD) are respectively of SEQ IDNOs: 8 and 9.

(Genetically Manipulated Cells)

When the DNA introduction system or the DNA introduction method of thepresent invention is carried out, a genetically manipulated cell isgenerated. Therefore, the present invention also provides a geneticallymanipulated cell thus obtained. The cell of the invention can exert newcharacteristics and functions as a result of the genetic manipulation.Such a cell can be intended for use in production of specificsubstances, treatments of specific diseases, and the like, according tointroduced DNA. In addition, the cell is also useful as a researchmaterial to examine the functions of the introduced DNA.

(DNA Introducing Kit)

The present invention further provides a DNA introducing kit used in theDNA introduction system and the DNA introduction method of the presentinvention. The DNA introducing kit has a donor factor as a transporterof desired DNA and a helper factor as a transposase source as essentialconstituent factors. Specifically, the donor factor is made of anexpression construct containing Tol1 element in which a polynucleotideencoding a transposase is defected and having an insertion site.Meanwhile, the helper factor is made of an expression constructcontaining the transposase of the present invention or a polynucleotideencoding the transposase. The “insertion site” herein means a site inwhich an objective DNA to be introduced is inserted. A restrictionenzyme recognition site inherent in Tol1 element can be utilized as the“insertion site.” For example, Tol1 element depicted in the basesequence of SEQ ID NO: 10 (Tol1-tyr, 1855 base pairs, not containingTSD) has a SalI site, and the restriction enzyme recognition site can beused as an insertion site. A restriction enzyme recognition site and abase sequence for a recombination reaction may be produced by a geneticengineering technique to use these as insertion sites. The base sequencefor a recombination reaction refers to an attR sequence used in, forexample, Gateway (registered trademark, Invitrogen Co.) technology.

Different kinds of plural restriction enzyme recognition sites may beformed as insertion sites. That is, a donor factor having amulti-cloning site (MCS) may be used. Although the kind of eachrestriction enzyme recognition site constituting a multi-cloning site isnot particularly limited, it is preferable to employ frequently usedrestriction enzyme recognition sites such as HindIII, BamHI, and EcoRI.This is because a kit with high versatility is constructed with thesesites. In addition, the donor factor (pDon253Mcs) shown in examplesdescribed later has multi-cloning sites made of bamHI, EcoRI, EcoRV,KpnI, PstI, XbaI, etc.

As described above, it was revealed that the transposition efficiency isnot damaged also when internal 1592 base pairs (from 158th base to1749th base counting from the 5′-end) of Tol1-tyr are removed. Based onthis finding, one embodiment of the present invention uses Tol1 elementhaving a structure in which insertion sites are formed between the5′-end side DNA and the 3′-end side DNA, obtained by removing at leastfrom the 158th base to the 1749th base counting from the 5′-end in thebase sequence of Tol1-tyr (base sequence of SEQ ID NO: 10). In otherwords, the embodiment uses a donor factor having a structure in whichdesired DNA is inserted between DNA in the 5′ end region (157 base pairsat maximum length) of the base sequence of SEQ ID NO: 10 and DNA in the3′ end region (106 base pairs at maximum length) of the base sequence ofSEQ ID NO: 10. A specific example of the 5′-end side DNA is DNA havingthe base sequence of SEQ ID NO: 21 (157 base pairs), and a specificexample of the 3′-end side DNA is DNA having the base sequence of SEQ IDNO: 22 (106 base pairs).

In a preferable embodiment, a vector containing Tol1 element in which apolynucleotide encoding a transposase is defected and having aninsertion site is used as the donor factor, and a vector containing apolynucleotide (the polynucleotide of the present invention) encoding atransposase is used as the helper factor. Such a kit has highconvenience, and a high DNA introduction efficiency can be expected fromits use. The helper factor in this case further contains a promoteroperably linked to a polynucleotide encoding a transposase and/or apoly-A additional signal sequence or a poly-A sequence connected to thepolynucleotide in the downstream side.

(Reconstructed Transposon)

A further aspect of the present invention provides a reconstructedtransposon. The reconstructed transposon of the present invention has astructure in which a polynucleotide encoding a transposase (that is, thepolynucleotide of the present invention) is inserted in Tol1 element inwhich polynucleotide encoding a transposase is defected. Preferably, apromoter is also inserted in Tol1 element so as to be operably linked tothe polynucleotide encoding a transposase. Not but that insertion of apromoter is not indispensable, and when the inserted “polynucleotideencoding a transposase” contains a sequence of a promoter region andsufficient transcription activity can be obtained by itself, insertionof the promoter can be omitted. On the other hand, it is preferable thata poly-A additional signal sequence or a poly-A sequence is alsoinserted in order to enhance stability of a transcription product(mRNA). That is, in one preferable embodiment of the reconstructedtransposon of the present invention, a poly-A additional signal sequenceor a poly-A sequence is connected to a polynucleotide encoding atransposase in the downstream side.

An insertion operation of a polynucleotide encoding a transposase, andthe like, may be carried out following a conventional method (seeMolecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press,New York), Current protocols in molecular biology (edited by FrederickM. Ausubel et al., 1987), etc.). Further, for terms such as “operablylinked,” and “promoter,” the above description (section of expressionconstruct containing a Tol1 element transposase) can be referred to.

A specific example of a reconstructed transposon is one having asequence in which the base sequence of SEQ ID NO: 3 or SEQ ID NO: 4 isinserted in the base sequence of any of SEQ ID NOs: 10 to 12.

The reconstructed transposon contains a transposase in a state ofcapable of expressing in a target cell and functions as an autonomoustransposable element. Therefore, the reconstructed transposon can beused as a tool for introducing DNA independently. The present inventionthus also provides a DNA introduction system using a reconstructedtransposon. “Independently” mentioned herein means that there is no needto use a separately prepared transposase in combination, and does notexclude combination use of components and elements (such as vectorskeletons and reagents) required for exerting the functions of thereconstructed transposon.

Matters not particularly mentioned in the present specification (such asconditions and operational methods) may be selected followingconventional methods, and for example, Molecular Cloning (Third Edition,Cold Spring Harbor Laboratory Press, New York), Current protocols inmolecular biology (edited by Frederick M. Ausubel et al., 1987), etc.can be referred to.

Example 1 1. Materials and Methods (1) Fish

Complete albino phenotype medaka fish were found from a commerciallybred group more than 30 years ago (Cited document 27). A line wasestablished from this individual, and kept in an experimentallaboratory. In this albino mutant, 1.9 kb of Tol1 element is inserted inthe first exon of a tyrosinase gene (Cited document 11). An individualshowing mosaic pigmentation appeared in a subline maintained in NiigataUniversity in 2001. Pigmentation did not occur in the primary linemaintained in Nagoya University. The primary line was determined to calli¹-Tomita and the pigmented subline was determined to call i¹-Niigata(Cited document 12). These designations are simplified and expressed asthe subline A and the subline B, respectively. Both of the sublines havenever been hybridized with other lines so far.

(2) Database

The following databases provided for public use were used: database usedfor constructing a base sequence that is considered to be full-lengthTol1 element: genomic project of medaka fish(http://shigen.lab.nig.ac.jp/medaka/genome/); database used forsearching a motif: MOTIF (http://motif.genome.jp/); database used forcollecting sequences of transfer enzymes of the hAT family: Pfam(http://www.sanger.ac.uk/Software/Pfam/); and database used for checkingamino acid sequences: Clustal X(http://bips.u-strasbg.fr/fr/Documentation/ClustalX/).

(3) Reagents and Kits

The following reagents and kits for molecular biology were used inaccordance with instructions of manufacturers: DNA amplification in PCR:PCR enzyme ExTaq (Takara Bio Inc., Otsu, Japan); production of genomiclibrary: fosmid vector pCC1FOS (EPICENTRE Biotechnologies, Madison,USA); labeling and hybridization analysis of probes: AlkPhos DirectLabelling and Hybridization System (GE Healthcare, Chalfont St. Giles,UK); RNA extraction: RNeasy kit (OA GEN GmbH, Hilden, Germany); RACEanalysis: FirstChoice RLM-RACE kit (Ambion, Austin, USA); incorporationof DNA into cells: PolyFect Transfection Reagent (QIAGEN GmbH); andselection of G418-resistant cells: G418 (Invitrogen Corp., Carlsbad,USA). Experimental conditions are described in the section ofexperimental results and the section of description of the drawings.

(4) Analysis of Transposition in Mammal Cultured Cells

Human HeLa cells and mouse NIH/3T3 cells were used. The cells weremaintained in a thermostat containing 5.0% of CO₂ at 37° C. using a DMEMculture medium containing a 10% bovine serum and an antibiotic.

1×10⁵ cells were seeded per one 35 mm-dish, and the temperature was keptfor 24 hours. A mixture of plasmid DNA was adjusted to 1000 ng per onedish and allowed to be incorporated into the cells using a PolyFectreagent. The temperature was kept for further 24 hours, the cells werethen washed twice with PBS, and a new culture medium without containingplasmid DNA and an incorporation reagent was added to keep thetemperature. After 24 hours, the cells were separated from the bottom ofthe dish with a trypsin treatment and suspended in 2.0 ml of the culturemedium. 100 μl each of the suspension was transferred to dishes ofdifferent sizes (35 mm, 60 mm, 90 mm). G418 was added to the culturemedium so as to make the volume of the medium 500 μg/ml. After selectionin G418 was continued for 12 days, the cells were fixed with 20%formalin and stained with a Giemsa stain solution. A dish having thecolony number closest to 100 was selected and the number of colonies wascounted. From the result, the colony number per 10⁵ cells initiallyseeded was estimated. The above-described analyses were all carried outby simultaneously preparing three groups of measurement systems.

2. Experimental Results (1) Modification of Fish as Material

The ratio of pigmented individuals among all individuals, that is, thepenetrance of pigmentation was 20% in the mosaic pigmented linediscovered in 2001. In order to modify the line into a material suitablefor a molecular level analysis, operations of selecting each one of maleand female fish having thick pigmentation to breed were performed overfive generations. As a result, the penetrance was 90% or more and alsoblack spots became larger (FIG. 1).

(2) Construction of Sequence Considered to be Full-Length Tol1 ElementFrom Data Analysis

A copy of Tol1 element (Tol1-tyr) initially isolated as insertion intothe inside of a tyrosinase gene has a length of 1.9 kb (SEQ ID NO: 10,containing no TSD). Inverted repeat sequences are respectively presentin the 5′-end side and the 3′-end side of Tol1-tyr. The sequence in theinverted repeat sequence in the 5′ end region of a sense strand(direction from 5′-end to 3′-end) is of SEQ ID NO: 5 and, in the samemanner, the inverted repeat sequence in the 3′ end region of a sensestrand (direction from 5′-end to 3′-end) is of SEQ ID NO: 6. Anonautonomous copy is formed due to deletion of the inside in a DNA typeelement in many cases (Cited document 19). Operation of searching alonger copy based on Tol1-tyr from a base sequence database was repeatedin consideration of such a fact. In each search, when a plurality ofcombinations of a sequence as the base with a newly added sequence werefound in the database, these combinations were regarded as a part offull-length Tol1. Based on the combinations, next search was thenperformed. While the operation was repeated, a portion that was notpresent in Tol1-tyr was gradually grown to be finally a 4.3 kb sequence.Then, this 4.3 kb sequence was anew checked with the database. For aposition of each base of 4.3 kb, a base having the highest appearancefrequency was adopted. As a result, a 4.3 kb sequence having 2.3 kb ofan open reading frame (ORF) was obtained. This sequence is expressed asTol1-L0.

(3) Identification of Autonomous Tol1 Element

An operation of amplifying an internal 1.2 kb portion of the sequence ofTol1-L0 (part of FIG. 2 b) from genomic DNA of a mosaic pigmentedsubline (expressed as fish B) by PCR was performed. A fragment obtainedby the amplification was used as a probe, and colony hybridization for agenomic library of fish B was carried out to obtain two clones. Theseclones are called Tol1-L1 and Tol1-L2. These two clones both have 4.3kb, and there was no difference observed in a restriction enzyme mapformed using five types of restriction enzymes (data not shown). Thus, abase sequence was examined only using Tol1-L1. As a result, the basesequence (SEQ ID NO: 4) and structure of Tol1-L1 were determined (FIG.2). Information from a further analysis described below and comparisonbetween structures of Tol1-L1 and Tol1-tyr are also shown in FIG. 2.

(4) Identification of Full-Length cDNA Considered to Encode Tol1Transposase

3′ RACE (rapid amplification of cDNA ends) for identifying atranscription product from a Tol1 transposase gene was performed usingRNA extracted from an albino line without pigmentation (fish A) and amosaic pigmented subline (fish B). As a result of analyzing amplifiedproducts by southern blotting, one signal was observed in fish B and nosignal was observed in fish A (FIG. 3). The result indicates that a Tol1transcription product from ORF exists in fish B but does not exist infish A, or even if it exists, the amount thereof is very small.Subsequently, 5′ RACE of fish B was performed and it was confirmed thatone signal appeared (FIG. 3). When clones of the RACE products causingthese signals were obtained and the base sequences were examined, thecDNA sequence (SEQ ID NO: 3) with 2.9 kb having ORF (SEQ ID NO: 1) wasobtained (FIGS. 11 to 13). This sequence was registered inDDBJ/EMBL/GenBank. The Accession No. is AB264112. By comparing thesequence of full-length cDNA and the Tol1-L1 sequence (SEQ ID NO: 4), itwas revealed that a Tol1 transposase gene is composed of three exons(FIG. 2).

When BLAST search was performed using the amino acid sequence deducedfrom Tol1 ORF (SEQ ID NO: 2, FIGS. 11 to 13), a list mainly composed oftransposable elements in the hAT family was formed. Elements having thehighest similarity among listed sequences were hAT family elements ofrice and Arabidopsis (data not shown). Further, when a motif of an aminoacid sequence was searched, presence of the dimerization domain(PF05699) of the hAT family registered in the Pfam database wasexpected. Further, by checking amino acid sequences of Tol1 and hATelements of various biological species, amino acid blocks preserved inthis family were confirmed to be present in Tol1 ORF (FIG. 4).Similarity of the amino acid sequence to Tol2 was lower than similarityto elements included in FIG. 4 (data not shown).

(5) Structure of Tol1 Copy Present in Medaka Fish Genome

The database search previously performed suggested that presencefrequency of a Tol1 internal region is less than that of an end regionin medaka fish genomes. This suggestion was confirmed by a southern blotanalysis on various kinds of medaka fish lines. Probes used correspondedto various parts of full-length Tol1-L1. When a probe for an end regionwas used, 100 bands or more appeared; on the other hand, when a probefor a central region was used, the number of bands was 0 to 5 (FIG. 5).Such a phenomenon is commonly observed also in Activator element of corn(Cited document 19), p element of drosophila (Cited document 20), andother DNA type elements. Broadly accepted description for thisphenomenon is that internal deletion is a main structure for generatinga nonautonomous element from an autonomous element (Cited documents 19and 20). This description may also be applicable to Tol1.

(6) Demonstration of Tol1 Transposition in Mammal Cells

In order to examine whether Tol1 ORF encodes a transposase and thetransposase has a function of intervening Tol1 element transposition ornot, first, a donor plasmid (hereinafter also referred to as a “donor”)and a helper plasmid (hereinafter also referred to as a “helper”) wereprepared. The donor plasmid had 1.9 kb of Tol1-tyr, and aneomycin-resistant gene was incorporated in the Tol1-tyr inside. Thehelper plasmid had Tol1 ORF. A CMV promoter for control was connected tothe upstream side of the ORF, and in the same manner, a poly-Aadditional signal was connected to the downstream side for stabilization(FIG. 6). A defective helper plasmid for a negative control wasprepared. The defective helper plasmid was obtained by modifying twocodons of the internal ORF into termination codons. Further, a fillerplasmid having an irrelevant DNA fragment with the same length as Tol1ORF was prepared. The filler plasmid was used to attain a constant DNAincorporation efficiency by making the whole DNA amount constant in agene introduction experiment. These plasmids were combined to introduceinto human HeLa cells and mouse NIH/3T3 cells, and cells that acquiredG418 resistance were selected. A large number of G418-resistant colonieswere generated in cells introduced with a donor and a helper, comparedwith the case of introducing a donor and a defective helper or the caseof introducing a donor and a filler (FIG. 7). In order to confirm thatacquisition of resistance is a result of transposition (incorporation)of Tol1 into genomic DNA of a host cell, a DNA fragment containing Tol1was cloned from a resistance-acquired cell obtained when introducing adonor and a helper, and base sequences of a Tol1 end region and aflanking portion were examined. When eight clones were examined,sequences in adjacent portions were all different (FIG. 8). 8 bp oftarget site duplications (TSD) were also found in all clones. Thisindicates that incorporation of a Tol1 portion of the donor plasmid intoa chromosome is a result of the transfer reaction. From the aboveresults, it was proved that Tol1 ORF encodes a functional Tol1transposase.

(7) Comparison Between Transposition Frequencies of Tol1 and Tol2

Transposition frequencies of Tol1 and Tol2 were examined using HeLacells. A donor and a helper respectively corresponding to Tol1 and Tol2were prepared and co-introduced into the HeRa cells in a predeterminedintroduction amount. Amount ratios of the donor and the helper were setwithin the range of 1:0.5 to 1:9 (in the case where the amount of donoris 100 ng) and within the range of 1:0.5 to 1:4 (in the case where theamount of donor is 200 ng). Within these ranges, transpositionfrequencies for both elements showed positive correlation to the amountof the helper (FIG. 9). The “net colony number” can be found bysubtracting the “colony number with no helper” from an “observationvalue of the colony number.” The maximum “net colony number” of Tol1 was3780−120=3660 (donor 200 ng, helper 800 ng) and that of Tol2 was3,393−287=3,106 (donor 200 ng, helper 400 ng). The ratio of maximumvalues (Tol1 maximum value/Tol2 maximum value) was 1.18. Thus, themaximum values of transposition frequencies of Tol1 and Tol2 wereequivalent.

(8) Noninterference Regarding Transposition Induction of Tol1 and To12

Both of Tol1 and Tol2 are hAT family elements, and also the elements arepresent in genomes of the same fish species. Thus, it was examinedwhether a Tol1 transposase induces Tol2 transposition or not and whethera Tol2 transposase induces Tol1 transposition or not. In thisexperiment, the ratio of a donor and a helper was set to 1:4 to beintroduced into HeLa cells. It was shown in past experiments that theratio causes transposition at a high frequency.

The experimental result clearly showed that the Tol1 transposase doesnot induce Tol2 transposition and the Tol2 transposase does not induceTol1 transposition (FIG. 10). Thus, it was revealed that these two typesof transposases respectively have functions specialized in correspondingelements.

3. Discussion

Two types of transcription factors have been conventionally known astransposable elements that are present in genomes of vertebrates andhave transposable activities. The transcription factors are Tzf elementof zebrafish and Tol2 element of medaka fish. Tol1 element had beenfound as insertion to be a cause of mutation of the albino line (sublineA) of medaka fish before these elements were found (Cited document 21),however, transposition of Tol1 element was not directly demonstrated atthe time of discovery. The reason is speculated that internal disruptionor deletion occurs in almost all of copies found at that time and othercopies that would be present in genomes. The present inventorsdemonstrated at this time that complete Tol1 element that can causetranscription at a high frequency is present through a database analysisand an analysis on the subline (subline B) having a unique character ofmosaic pigmentation.

Tol1 and Tol2 both belong to the hAT family. However, there are largedifferences in structures on a molecular level and distribution amongspecies. A large number of copies of Tol1 have internal deletion ofvarious sizes and repeat sequences having homology in Tol1 are widelydistributed in the Oryzias (Cited document 10). Contrary to the Tol1copies, Tol2 copies have uniform structures even on a base sequencelevel and are observed only in medaka fish (O. latipes) and its relatedspecies (O. curvinotus) (Cited document 23). The inventors speculatefrom such situations that Tol1 is an element that has been present inthe Oryzias from a long time ago; on the other hand, Tol2 is an elementthat recently invaded in a line related to medaka fish (Cited document23). It is considered that these two elements only coexist in currentmedaka fish by chance, and a sufficiently long time have passed so far;therefore, separate transfer reaction lines have been established bynow.

Mosaic pigmentation caused in excision of Tol1-tyr is similar to aphenomenon of unstable body color mutation that was recently discoveredin another allelic gene by the present inventors (Cited document 9). Ina line showing unstable body color mutation, excision of Tol2 from atyrosinase gene is caused at high frequency, and in addition, Tol2insertions occur in various sites of a genome. Although bursting ofactive transposition by a transposable element, that is, transpositionburst, is frequently observed in model organisms other than vertebrates,the phenomenon of Tol2 of medaka fish was the first example oftransposition burst in vertebrates. Examination of insertion of Tol1 isdifficult at present. This is because the number of Tol1 copies inmedaka fish genomes is far larger as compared with that of Tol2, andthus is beyond the limit of the current analysis method. Iftransposition burst also occurs in Tol1, there is a possibility that aDNA type element gives a great influence on genomic evolution ofvertebrates, and thus reexamination regarding the degree of theinfluence is required.

It had been considered that Tol1 is an element that has already lost itsfunctions until mosaic pigmented medaka fish was discovered. The reasonis that, even though the number of Tol1 copies exceeding 100 wasexamined, no structure assumed to be a gene was found (Cited document10). It was revealed from the results of human and other genomicprojects that a considerable amount of DNA transposable elements existin genomes of vertebrates. However, most of the elements have losttransposable activities (Cited document 4). The results at this time bythe inventors present a question whether an element capable ofreactivation exits among these DNA transposable elements or not. Inparticular, the result of southern blotting that a potential autonomouscopy exists in fish A has a significant meaning.

In a comparison experiment of transposition frequencies of Tol1 andTol2, amounts of the donor and the helper to be introduced were varied.The plasmid amount was set on the weight basis at this time. As aresult, the ratio of molar numbers of the plasmids used for introductionof Tol1 and Tol2, that is, the ratio of molecular numbers was not thesame. This is because the size of a transposable element in the donorand the size of cDNA in the helper are different between the twoelements. In the case of the donor plasmid, the sizes of the wholeelements containing neomycin-resistant genes were 3.7 kb in Tol1 and 3.1kb in Tol2. The sizes of encoding regions in the helper plasmid were 2.8kb in Tol1 and 2.0 kb in Tol2 (see descriptions of FIGS. 5 and 9 fordetails). Although there are such differences, comparative transpositionefficiencies of Tol1 and Tol2 can be read out from the experimentalresult. A particularly important point is that the maximum value of theTol1 transposition frequency was equal to the maximum value of the Tol2transposition frequency. Tol2 is an element recently used in developinggenetic modification systems for vertebrates such as gene introduction(Cited document 13), mutagenesis (Cited document 14), and trapping ofgenes and promoters or enhancers (Cited document 15). Accordingly, itcan be considered that Tol1 holds similar possibilities. In thiscontext, the fact that two types of elements give no influence ontransposition each other is extremely important. Existence of two DNAintroduction systems capable of independent control is particularlybeneficial in a situation where continuous introduction of two types ofDNAs into one cell line or organism is necessary. It is also envisagedthat one of the two types of DNAs after introduction is transferred bysupplying a transposase of an element corresponding to the DNA as oneusage form of these two DNA introduction systems.

While both of Tol1 and Tol2 are elements in the hAT family, SleepingBeauty and Frog Prince which have been reconstructed by DNA moleculeprocess belong to the mariner/Tc1 family. piggyBac derived from insectsbelongs to another type. A significant difference recognized among thesetransposable element groups lies in the size of an element. Most of themariner/Tc1 family elements have 1 to 2 kb, and piggyBac has 2.5 kb. Onthe other hand, a typical full-length element in the hAT family has 4 to6 kb. Since negative correlation exists between element sizes andtransposition frequencies in many cases (Cited document 24), having alarge size of an element belonging to the hAT family would be a usefulproperty to transfer a large-sized DNA fragment. In fact, the presentinventors reported that Tol2 having a size as large as even 9.0 kb canbe transferred (Cited document 13). Other than the element size, thereis an important point as a difference recognized among the transposableelement groups. The difference is the way of appearance of “restrictionaccompanied by excessive expression.” When a transposase exists in anexcess amount, transposition frequencies are known to decrease inSleeping Beauty (Cited document 24), mariner of drosophila (Citeddocument 25), and piggyBac (Cited document 25). However, such aphenomenon did not appear in this experiment on Tol1 and Tol2. In thesame manner, the phenomenon was not recognized also in another study onTol2 (Cited document 26). Such an achievement that two hAT familyelements, which independently function and have high transpositionfrequencies, can be used leads to extension and development of geneticmanipulation techniques Intended for vertebrates.

CITED DOCUMENTS

-   1. Dombroski B. A., Mathias S. L., Nanthakumar E., Scott A. F.,    Kazazian H. H., Jr (1991) Science 254: 1805-1808.-   2. Burden A. F., Manley N. C., Clark A. D., Gartler S. M., Laird C.    D., Hansen R. S. (2005) J Biol Chem 280: 14413-14419.-   3. Li X., Scaringe W. A., Hill K. A., Roberts S., Mengos A., Careri    D., Pinto M. T., Kasper C. K., Sommer S. S. (2001) Hum Mutat 17:    511-519.-   4. International Human Genome Sequencing Consortium (2001) Nature    409: 860-921.-   5. Lam W. L., Lee T. S., Gilbert W. (1996) ProC Natl Acad Sci USA    93: 10870-10875.-   6. Koga A., Suzuki M., Inagaki H., Bessho Y., Hori H. (1996) Nature    383: 30.-   7. Bryan G., Garza D., Hartl D. L. (1990) Genetics 125: 103-114.-   8. Brookfield J. F. (2004) Curr Biol 14: R344-345.-   9. Koga A., Tida A., Hori H., Shimada A., Shima A. (2006) Mol Biol    Evol 23: 1414-1419.-   10. Koga A., Sakaizumi M., Hori H. (2002) Zoolog Sci 19: 1-6.-   11. Koga A., Inagaki H., Bessho Y., Hori H. (1995) Mol Gen Genet    249: 400-405.-   12. Tsutsumi M., Imai S., Kyono-Hamaguchi Y., Hamaguchi S., Koga A.,    Hori H. (2006) Pigment Cell Res 19: 243-247.-   13. Koga A., Hori H., Sakaizumi M. (2002) Mar Biotechnol 4: 6-11.-   14. Johnson Hamlet M. R., Yergeau D. A., Kuliyev E., Takeda M.,    Taira M., Kawakami K., Mead P. E. (2006) Genesis 44: 438-445.-   15. Choo B. G., Kondrichin I., Parinov S., Emelyanov A., Go W.,    Toh W. C., Korzh V. (2006) BMC Dev Biol 6: 5.-   16. Ivics Z., Hackett P. B., Plasterk R. H., Izsvak Z. (1997) Cell    91: 501-510.-   17. Miskey C., Izsvak Z., Plasterk R. H., Ivies Z. (2003) Nucleic    Acids Res 31: 6873-6881.-   18. Wu S. C., Meir Y. J., Coates C. J., Handler A. M., Pelczar P.,    Moisyadi S., Kaminski J. M. (2006) ProC Natl Acad Sci USA 103:    15008-15013.-   19. Rubin E., Levy A. A. (1997) Mol Cell Biol 17: 6294-6302.-   20. O'Hare K., Rubin G. M. (1983) Cell 34: 25-35.-   21. Koga A., Hori H. (1997) Pigment Cell Res 10:377-831.-   22. Koga A., Hori H. (1999) Genet Res 73: 7-14.-   23. Koga A., Shimada A., Shima A., Sakaizumi M., Tachida H.,    Hori H. (2000) Genetics 155: 273-281.-   24. Geurts A. M., Yang Y., Clark K. J., Liu G., Cui Z., Dupuy A. J.,    Bell J. B., Largaespada D. A., Hackett P. B. (2003) Mol Ther 8:    108-117.-   25. Hartl D. L., Lozovskaya E. R., Nurminsky D. I.,    Lohe A. R. (1997) Trends Genet 13: 197-201.-   26. Kawakami K., Noda T. (2004) Genetics 166: 895-899.-   27. Tomita H. (1975) in Medaka (Killifish): Biology and Strains, ed    Yamamoto T. (Yugakusha Publ., Tokyo), pp. 251-272.-   28. Rubin E., Lithwick G., Levy A. A. (2001) Genetics 158: 949-957.

Example 2

Now that an autonomous copy of Tol1 element (Tol-L1, length 4355 bp,DDBJ AB288091, SEQ ID NO: 4) and a transposable enzyme gene (length 2900bp, DDBJ AB264112, SEQ ID NO: 3) are in hand as clones (Example 1), itcan be thus recognized that Tol1 element is available as a genetic toolapplicable to mammals.

Reduction of the transposition frequency with decrease of the size of anelement is a common feature of transposable elements. Thus, “loadingability” is important when an element in use is selected. The “loadingability” herein means the “maximum length of a DNA fragment that can becarried by an element.” Tol1 element is expected to be highly useful inthis regard. The first reason is that Tol1 element belongs to the hATfamily. The hAT family is a group of transposable elements representedby hobo element of drosophila, Activator element of corn, and Tam3element of snapdragon (Cited documents 2 and 16). A particularcharacteristic of this family is a longer complete copy as compared withother main families. Specifically, while the hAT family elements havelengths of 4 to 6 kb, most elements in the mariner/Tc1 family have 1 to2 kb and piggyBAac element has 2.5 kb, which are comparatively short.The second reason is due to the result of a preliminary examination bythe present inventors preceding this study. That is, it was suggestedthat Tol1 element exceeding 15 kb exists in medaka fish genomes. Thepresent inventors inferred based on these findings that Tol1 elementtransfers even if the whole length thereof exceeds 15 kb. Tol2 elementis also a hAT family element (Cited document 7). However, copies of thiselement have no difference in structures thereof, and almost all copieshave 4.7 kb. Although the present inventors have made large scaleinvestigations on naturally existing Tol2 element so far, an elementexceeding 4.7 kb has not been found (Cited documents 7 and 8).

In this study, the length of naturally existing Tol1 element wasinvestigated. As a result, it was revealed that copies having lengths ofabout 18 kb and about 20 kb exist. Then, “development of a geneintroduction vector having an ability to incorporate long DNA into achromosome,” which is the main object of this study, was carried out.First, an internal region unnecessary for a transfer reaction wasremoved from an element with 1.9 kb to prepare a short vector having awhole length of 0.3 kb. Then, another DNA fragment was inserted in thisvector that is the base to prepare Tol1 elements with various lengths.Each Tol1 element was then incorporated in a cell by a lipofectionmethod. Subsequently, selective culture was performed with an antibioticG418, thereafter the residual colony number was counted to calculate atransposition frequency (see Example 1 for the experimental technique).However, since it was concerned that the DNA incorporation efficiency bythe lipofection method might give an influence on the final results, acountermeasure for excluding the influence of the incorporationefficiency was taken. That is, the countermeasure is to make acomparison after making sizes of whole plasmids uniform even if lengthsof Tol1 elements are different. The obtained results revealed that evenwhen Tol1 element has such a long whole length as 22.1 kb, the Tol1element efficiently transfers. The length is the longest as a reportedvalue as compared with other DNA transposable elements used in mammals.

1. Materials and Methods (1) Genomic Library

A genomic library of medaka fish was prepared in a previous study (Citeddocument 10). The library was used in order to obtain a clone of genomicDNA containing Tol1 element in this study. The original genomic DNA ofmedaka fish for the library was extracted from albino medaka fish havingpartial melanin pigmentation on its skin and eyes. The vector was fosmidpCC1FOS (EPICENTRE Biotechnologies, Madison, Wis., USA), which containsmechanically sheared DNA with 33 to 48 kb in its inside.

(2) Plasmid

Two types of plasmids, i.e. a donor and a helper, were used. Tol1element was cut out from the donor and incorporated in a chromosome dueto an action of a transfer enzyme made from the helper in a cell.

The structure of the donor used at this time is shown in FIGS. 15 and17. The helper was the same as that used in Example 1, and the basicstructure thereof is shown in FIG. 15. A defective helper was preparedin addition to a complete helper. The defective helper serves as acontrol section involved with an action of a transfer enzyme.

(3) Transposition Frequency Measurement System

Human HeLa cells and Mouse NIH/3T3 cells were cultured in a DMEM mediumcontaining 10% FBS. The culture temperature was set to 37° C. and theCO₂ concentration was set to 5.0%.

2×10⁵ cells were seeded per one well in a 12-well plate (diameter 22mm). After 24 hours, 100 ng of the donor and 900 ng of the helper wereallowed to be incorporated into the cells using a Lipofectamine LTXreagent (Invitrogen Corp., Carlsbad, N. Mex., USA). After 8 hours, thecells were washed twice with PBS and the medium was replaced with a newone. After 24 hours, the cells were separated from the dish with trypsinand suspended in 2.0 ml of a medium. Mediums containing G418 at aconcentration of 500 μg/ml were placed in dishes of different sizes (35mm, 60 mm, and 90 mm), and 400 μl of a suspension was subsequentlycharged thereto. The procedure from this operation is culture forselection with G418. After selective culture for 12 days, cells werefixed with 20% formalin and stained with a Giemsa stain solution. A dishhaving the colony number closest to 100 was selected among the disheswith three kinds of sizes and the colony number was counted. The colonynumber per 10⁵ cells initially seeded was found based on the number anda dilution ratio. The above-described procedure is a process of onetrial. This trial was performed three times per each combination of thedonor and the helper.

(4) Technique of Molecular Level Operation

This study is extension of the study shown in Example 1. Adjustment ofgenomic DNA, PCR, cloning of PCR products, determination of basesequences, and colony hybridization were carried out by the same methodsand procedures. However, for an enzyme for PCR, LA Taq (Takara Bio Inc.)that efficiently performs amplification of long DNA was used, instead ofEx Teq (Takara Bio Inc., Otsu, Japan) used in the previous time. Theconditions of PCR are described in the corresponding section.

2. Experimental Results (1) Mutation of Length Shown in Tol1 Copies

100 to 200 copies of Tol1 exist in a genome of medaka fish, and lengthsthereof are not uniform (Cited document 9). In order to know a degree ofmutation of a length, screening of a genomic library was performed withparticular attention to identification of long copies. Twohybridizations were carried out in the screening and a chromosomefragment containing both end regions of Tol1 was recovered. In the firstscreening, a clone hybridized with the left end region (1st to 500thbases of SEQ ID NO: 10) of Tol1-tyr was selected. The colony numberintended for screening was 4×10⁴ (corresponding to an about doubled DNAamount of a haploid genome) and a probe labeled with alkalinephosphatase was used. In this screening, 161 positive signals weredetected. Subsequently, the second screening was performed for a colonycorresponding to each positive signal. The region used for a probe atthis time was a right end region of Tol1-tyr (1356th to 1855th bases ofSEQ ID NO: 10). In this screening, 130 out of 161 colonies wereselected.

The clones obtained by the screening were subjected to PCR in which theboth end regions of Tol1 were used as primers. This is to amplify a Tol1portion contained in each clone. Amplification was observed in 114 outof 130 clones. The distribution of element lengths is approximatelymound-shaped and has a sharp peak at 1 to 2 kb (FIG. 16). It isparticularly notable that clones with 18 kb and 20 kb were found. Asshown later, Tol1 element transfers regardless of internal basesequences as long as it has end regions. Therefore, it is expected thattwo long Tol1 elements found at this time also have transferringability.

(2) Transposable Activity of Short Clones of Tol1

It is a frequently observed phenomenon that a DNA transposable elementdoes not lose a transposable activity as long as a transfer enzyme ispresent even though the element partially lacks the inside thereof(Cited document 15). This phenomenon would also be observed in Tol1.This is because Tol1-tyr element having 1.9 kb has a structure lackingthe inside of Tol1-L1 element having 4.4 kb and the Tol1-tyr elementtransfers due to supply of a transfer enzyme (Cited document 10). Sincea large number of elements shorter than 1.9 kb also exist in a medakafish genome (FIG. 16), it is considered that a portion unnecessary fortransposition still exists in this element with 1.9 kb. Based on thisassumption, a large number of shorter clones were prepared andtransposable activities thereof were respectively examined. The methodof measuring the number of the formed colonies in the same manner as inExample 1 was used. In preparation of shorter elements, a primerdesigned so as to be directed outward at an end region of Tol1-tyr wasused. First, an arm of Tol1 and a plasmid having the arm were amplifiedas one sequential fragment by PCR, and both ends of the fragment wereconnected. Then, a neomycin-resistant gene was incorporated in theconnected portion (FIG. 15). The donor plasmid thus obtained was allowedto be incorporated into cultured cells of a mouse together with acomplete or defective helper (FIG. 15). As a result, obtained was animportant finding such that a clone made of a left arm with 157 by andaright arm with 106 by showed a transposition frequency equal to or morethan that of Tol1-tyr (FIG. 16). The case of further deleting either ofarms in this clone to be 26 by resulted in a state where a transposableactivity is extremely decreased, and depending on cases, resulted inloss of a transposable activity (FIG. 16).

(3) Preparation of Short Vector Having Cloning Site

A new clone was prepared based on the previous experimental results. Thenew clone pDon263Mcs has 157 by of the left arm and 106 by of the rightarm of Tol1 element. The new clone has a cloning site (multiple cloningsite, MCS) corresponding to frequently used six types of restrictionenzymes (BamHI, EcoRI, EcoRV, KpnI, PstI and XbaI) between the both arms(FIG. 17). There is a HindIII site right outside Tol1 element. This siteis used for accurate measurement of a transposition frequency asdescribed in the following.

(4) Preparation of Plasmid Having a Constant Whole Length and HavingTol1 Element with Different Sizes in the Inside Thereof.

A DNA fragment with a length of x kb (x=0, 5, 10, 15, or 20) and anotherfragment with a length of y kb (y=20-x) were prepared utilizing PCR. Theformer was inserted in an EcoRI site of pDon263McsNeo (inside of Tol1)and the latter was inserted in the HindIII site (outside of Tol1) (FIG.17). Clones thus prepared were named pDon263McsNeoExHy. Regardinglengths of respective parts, a Tol1 arm was 0.3 kb, a neomycin-resistantgene was 1.8 kb, and a portion of a plasmid vector was 2.7 kb.Accordingly, the distance from the left end to the right end of Tol1 inpDon263McsNeoExHy was (x+2.1) kb. The size of the plasmid on the wholewas 24.8 kb regardless of the value of x.

It has been known that a plasmid size gives an influence on anincorporation efficiency at the time of DNA incorporation by alipofection method. A DNA fragment was also inserted in the outside ofTol1 in addition to the inside to uniformize sizes of the entireplasmids and the influence of sizes was thus excluded. Thereby, precisecomparison of transposition frequencies among donors with differentsizes becomes possible.

(5) Comparison of Transposition Frequencies

A transposition frequency of each of five types of donors was measured,combining the donors with a complete helper or a defective helper (FIG.18). Both inhuman and mouse cells, the size of an element and thetransposition frequency had negative correlation. Ratios oftransposition frequencies of the longest element (pDon263McsNeoE20) tothe shortest element (pDon263McsNeoH20) upon incorporation with acomplete helper were 0.21 in a human cell and 0.28 in a mouse cell. In ahuman cell, a transposition frequency upon combination with the completehelper in the case of using the longest element was 8 times as high asthat of the defective helper (10 times in a mouse cell).

(6) Demonstration of Transposition

Next, a trial was made to demonstrate that incorporation of Tol1 elementinto a chromosome is due to a transfer reaction. First, two colonies ofmouse cells obtained in the trial with the longest element(pDon263McsNeoE20) were isolated to establish lines. These lines (N1 andN2, N means a neomycin-resistant transformant herein) were respectivelyamplified and genomic DNA was extracted. The obtained DNA was used as atemplate and an end region of Tol1 and a chromosome region adjacent tothe end region were amplified. The amplification was carried out byinverse PCR. A base sequence of the obtained DNA fragment was thenexamined. It was found from the obtained base sequence that 8 bp of atarget site duplication was generated in both of the two cell lines(FIG. 19). Generation of target site duplication means that a reactionof incorporating a donor into DNA was a transfer reaction. It wasrevealed from BLAST search in the base sequence database of mice thatincorporated sites were chromosome 15 and chromosome 5.

Thus, incorporation into chromosomes by transposition was confirmed.However, it is unclear from these results that the whole Tol1 elementincluding an internal DNA fragment was incorporated in chromosomeswithout generating partial deletion or disruption. Therefore, a primercovering a Tol1 end region and a chromosome region adjacent to the endregion was prepared and PCR was performed for a further study. Thecultured cell was a diploid, and an attention was given to the fact thatinsertion of Tol1 was considered to occur only in one of the twohomologous chromosomes. The incorporated Tol1 element was amplified in aprimer designed as described above. It never happens that acorresponding site in the other chromosome is amplified. This is becausethe primer has a part of the base sequence of Tol1 element in the 3′-endand the part does not adapt to the corresponding site. Amplification wasonly observed for the case of a right combination of a cell line and aprimer in PCR (FIG. 20). The length of an amplified product was asexpected (22.1 kb) and an electrophoresis pattern obtained by cuttingthe amplified product with a restriction enzyme was also as expected(FIG. 20). It was demonstrated from these results that the whole regionof Tol1 element with 22.1 kb was incorporated into a chromosome by atransfer reaction without generating deletion or disruption.

3. Consideration (1) Mutation of Length of Tol1 Element

In this study, mutation of the length of Tol1 element was first examinedand it was found that copies with about 18 kb and about 20 kb exist in amedaka fish genome. This result indirectly supports assumption by thepresent inventors such that Tol1 element transfers even if the lengththereof exceeds 15 kb.

Methods used for examining mutation of the length were two screenings ofa genomic library and three PCRs on each clone. Other possible methodsinclude two methods of (1) analysis of a base sequence database ofmedaka fish and (2) PCR in which genomic DNA is directly used. However,the present inventors did not employ these methods. Sufficiency of thebase sequence database of medaka fish has been improving year by year.However, it still cannot be recognized that a long scattered repeatsequence such as a transposable element is precisely incorporated.Continuous sequences such as a contig or scaffold sequence are made byediting with a computer, and these sequences are often broken at theinside of long repeat sequences. Actually, autonomous Tol1 element with4.4 kb, which was identified in the previous study made by the presentinventors (Cited document 10), has not yet appeared in the database as atrain of one sequence (version 46 published in August 2007).

A method utilizing PCR that directly uses genomic DNA also cannot berecognized to be useful. The reason is that the number of copies havingsuch a short length as 1 to 2 kb are far larger than that of long copiesin Tol1 and the short copies are predominantly amplified in PCR. A largeamount of cloning and subsequent PCR analysis on each clone were onlyfeasible means in this study.

(2) Removal of Unnecessary Internal Region

The present inventors constructed a basic Tol1 vector made of the leftarm with 157 by and the right arm with 106 bp. The vector transferred ata high efficiency equal to that of the original element (1855 bp). Thepresent inventors thus succeeded in removing 1592 by of the internalregion. Due to this modification, a space for loading a DNA fragmentinside would have been increased. Further, this modification hassignificant meanings also in view of removal of a signal with apossibility of giving an influence on loaded DNA or a host cell and asequence similar to the signal.

It is considered that a more specific analysis enables an arm of avector to be cut shorter. Such an analysis was not performed in thisstudy. One of the reasons therefore lies in such an estimation thatremarkable increase of loading ability cannot be expected inconsideration of the fact that if either of the arms is cut to be 26 bp,the transposition frequency extremely decreases (FIG. 16) (the increasedcontent becomes (157−26)+(106−26)=211 by even at maximum). The otherreason is that if arms with certain lengths are preserved, the arms canbe used in an analysis of an element incorporated by transposition. Inmany cases, an initial operation of such an analysis is cloning of anadjacent chromosome region. The main technique thereof is inverse PCRand it is necessary to use a part of the arms as primer regions in theinverse PCR. Further, two times or more of nested type PCRs aresometimes necessary and this case results in use of different parts inthe arms as primers. Considering such a situation, the present inventorspreserved a portion that can be used for a PCR primer. The arm lengthsof the basic vector (157 by and 106 bp) were determined based on suchconsideration.

(3) Influence of Element Size on Transposition Frequency

Transposition frequencies were measured in the both cases of a completehelper and a defective helper. Formation of colonies was observed alsoin the case of the defective helper. However, as shown in Example 1,Tol1 element of a colony generated with the defective helper did notaccompany target site duplication; therefore, the colony was generatedby random insertion, not by transposition. The fact that the colonynumbers in the defective helpers were approximately the same among fivetypes of donors accords with this explanation. Further, this result alsoindicates that influence of a plasmid size on a DNA incorporationefficiency in a lipofection method was sufficiently excluded in themeasurement method used by the present inventors.

It was revealed from an analysis of transformant cells that the wholeregion of Tol1 element was incorporated into a chromosome by a transferreaction. The incorporation frequency was significantly high even in thecase of the longest Tol1 element (pDon263McsNeoE20) as compared with arandom incorporation frequency. The length of Tol1 element of this donorplasmid was 22.1 kb and 0.3 kb out of 22.1 kb was an arm of Tol1.Accordingly, the basic vector prepared at this time (pDon263Mcs) has anability of delivering DNA with a length up to 21.8 kb to a chromosome.It was also an important finding that the delivered DNA fragment did notcause internal deletion or disruption.

(4) Comparison with Other Transposable Elements

It has been known that Sleeping Beauty element loses a transpositionefficiency when the whole length exceeds 9.1 kb (Cited document 6). Ithas been found that piggyBac element functions as a gene introductionvector even when the whole length is 14.3 kb (Cited document 3). In thecase of Tol2 element, the maximum length reported so far is 10.2 kb(Cited document 1). Regarding only to piggyBac element and Tol2 element,there is a possibility that transposable activities are retained evenwhen lengths thereof are longer than the reported lengths. Currently,the value of 22.1 kb shown with Tol1 at this time by the presentinventors is the maximum among DNA transposable elements used inmammals. In addition, the basic vector prepared by the present inventorshas a portion used as an arm of Tol1 as short as 0.3 kb. From the abovedescription, it can be considered that Tol1 is a useful geneintroduction vector to allow long DNA to be incorporated in chromosomesof mammals.

CITED DOCUMENTS

-   1. Balciunas D., Wangensteen K. J., Wilber A., Bell J., Geurts A.,    Sivasubbu S., Wang X., Hackett P. B., Largaespada D. A., McIvor R.    S., Ekker S. C. (2006) Harnessing a high cargo-capacity transposon    for genetic applications in vertebrates. PLoS Genet 2: e169-   2. Calvi B. R., Hong T. J., Findley S. D., Gelbart W. M. (1991)    Evidence for a common evolutionary origin of inverted repeat    transposons in Drosophila and plants: hobo, Activator, and Tam3.    Cell 66:465-471-   3. Ding S., Wu X., Li G., Han M., Zhuang Y., Xu T. (2005) Efficient    transposition of the piggyBac (PB) transposon in mammalian cells and    mice. Cell 122: 473-483-   4. Fraser M. J., Ciszczon T., Flick T., Bauser C. (1996) Precise    excision of TTAA-specific lepidopteran transposons piggyBac (IFP2)    and tagalong (TFP3) from the baculovirus genome in cell lines from    two species of Lepidoptera. Insect Mol Biol 5: 141-151-   5. Ivics Z., Hackett P. B., Plasterk R. H., Izsvak Z. (1997)    Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon    from fish, and its transposition in human cells. Cell 91: 501-510-   6. Karsi A., Moav B., Hackett P., Liu Z. (2001) Effects of insert    size on transposition efficiency of the Sleeping Beauty transposon    in mouse cells. Mar Biotechnol 3: 241-245.-   7. Koga A., Hori H. (1999) Homogeneity in the structure of the    medaka fish transposable element Tol2. Genet Res 73: 7-14-   8. Koga A., Iida A., Hori H., Shimada A., Shima A. (2006) Vertebrate    DNA transposon as a natural mutator: the medaka fish Tol2 element    contributes to genetic variation without recognizable traces. Mol    Biol Evol 23: 1414-1419.-   9. Koga A., Inagaki H., Bessho Y., Hori H. (1995) Insertion of a    novel transposable element in the tyrosinase gene is responsible for    an albino mutation in the medaka fish, Oryzias latipes. Mol Gen    Genet 249: 400-405-   10. Koga A., Shimada A., Kuroki T., Hori H., Kusumi J.,    Kyono-Hamaguchi Y., Hamaguchi S. (2007) The Tol1 transposable    element of the medaka fish moves in human and mouse cells. J Hum    Genet 52: 628-635-   11. Koga A., Shimada A., Shima A., Sakaizumi M., Tachida H.,    Hori H. (2000) Evidence for recent invasion of the medaka fish    genome by the Tol2 transposable element. Genetics 55: 273-281-   12. Koga A., Suzuki M., Inagaki H., Bessho Y., Hori H. (1996)    Transposable element in fish. Nature 383: 30-   13. Koga A., Suzuki M., Maruyama Y., Tsutsumi M., Hori H. (1999)    Amino acid sequence of a putative transposase protein of the medaka    fish transposable element Tol2 deduced from mRNA nucleotide    sequences. FEBS Lett 461: 295-298-   14. Miskey C., Izsvak Z., Plasterk R. H., Ivics Z. (2003) The Frog    Prince: a reconstructed transposon from Rana pipiens with high    transpositional activity in vertebrate cells. Nucleic Acids Res 31:    6873-6881-   15. O'Hare K., Rubin G. M. (1983) Structures of P transposable    elements and their sites of insertion and excision in the Drosophila    melanogaster genome. Cell 34: 25-35-   16. Rubin E., Lithwick G., Levy A. A. (2001). Structure and    evolution of the hAT transposon superfamily. Genetics 158: 949-957-   17. Zagoraiou L., Drabek D., Alexaki S., Guy J. A., Klinakis A. G.,    Langeveld A., Skavdis G., Mamalaki C., Grosveld F.,    Savakis C. (2001) In vivo transposition of Minos, a Drosophila    mobile element, in mammalian tissues. Proc Natl Acad Sci USA 98:    11474-11478

Example 3

It was shown in Example 2 that Tol1 has a characteristic of being ableto carrying a long DNA fragment in a chromosome and is an excellentgenetic tool. Specifically, it was revealed that Tol1 effectivelytransfers even if the whole length is as long as 22.1 kb and that Tol1functions as a vector if the total length of left and right arms is only263 kb. Further, it has been already proved that Tol1 transfers inhumans and mice in addition to medaka fish (Example 1, Cited document15). Therefore, a transposable activity is expected to be present in awide range of vertebrates.

A DNA transposable element transfers mainly in a mode of “cut andpaste.” “Cut” indicates a process of drawing out an element from a DNAmolecule such as a chromosome on which the element is currently carried.“Paste” means incorporation of the element drawn out into the same oranother DNA. Herein, when “cut” occurs, detection thereof is easybecause sufficient information can be obtained by an analysis with PCRfocusing on a specific element. On the contrary, demonstration ofincorporation is not easy since it is impossible to know where theelement is to be incorporated in advance and a marker gene and acomplicated detection system are required.

An object of this study is to examine whether excision of toll elementis generated in X. laevis or not. For this object, an indicator plasmidin which Tol1 element is embedded and a helper plasmid for supplying atransfer enzyme in a cell were prepared. These plasmids were injected ina flog embryo in an initial developmental stage and recovered from theembryo after the elapse of a time for cell division. Subsequently, theindicator plasmid was analyzed by PCR, cloning, and base sequencemapping method. The result showed that excision of Tol1 element from theplasmid occurred. Various sequences as traces were also observed at abreaking point. From the above results, it was revealed that Tol1element shows transferring ability also in this model animal and, at thesame time, suggested that Tol1 has excellent versatility as a tool forgenomic manipulation.

The sequence of the trace was similar to those shown in fish andmammals. However, a tendency that a specific nucleotide appears at abreaking point was observed. Regarding this tendency, it can be alsoconsidered that some kinds of DNA repair mechanisms specific to thisfrog or amphibians exist, and the tendency reflects the mechanisms.

1. Materials and Methods (1) Plasmids

Two types of plasmids were used. They were an indicator and a helper.The former was a plasmid having a nonautonomous Tol1 element and thelatter was a plasmid having a transfer enzyme gene controlled by a CMVpromoter. It is expected that in cells, the helper supplies a transferenzyme, which catalyzes transposition of Tol1 element present in theindicator.

In addition to a complete helper, a defective helper was also preparedfor a control section of the functions of the transfer enzyme. This is ahelper obtained by changing two sites of codons corresponding to aminoacids in the middle of the transfer enzyme to termination codons.

Indicator plnd263GFP contains an arm of Tol1 element with 263 kb and aGFP gene. The complete helper pHel851aa encodes a transfer enzyme madeof 851 amino acids. The defective helper pHel316aa encodes a brokenprotein having only 316 amino acids. The structures are shown in FIG.21. The GFP gene in plnd263GFP is constituted with a CMV promoter, acoding sequence of EGFP, and a poly-A additional signal. This GFP geneserves as a marker gene to confirm that DNA injected into an embryo cellis incorporated in a nucleus.

(2) Injection of Frog Embryo and DNA

600 units and 300 units of chorionic gonadotropin (Aska Pharmaceutical,Tokyo, Japan) were respectively injected into a female frog and a malefrog, and the frogs were naturally bred to obtain a fertilized egg.After removing a jelly layer in 3% cysteine (pH 7.9) and washing thefertilized egg with 0.1× Steinberg's solution (Cited document 12), thefertilized egg was transferred to [3% FicoII, 0.1× Steinberg'ssolution]. When the fertilized egg became a four cell embryo from a twocell embryo, 5 nl of plasmid DNA was injected. DNA was dissolved in [88mM NaCl, 15 mM Tris-HCl (pH 8.0)] so that the concentration of theindicator would be 5 μg/ml and the concentration of the helper would be50 μg/ml. The embryo after completion of injection was cultured in 0.1×Steinberg's solution at 20° C. The ratio of the indicator to the helperwas 1:10, and the ratio was determined in reference to the result ofExample 1 (various ratios were set to examine transposition frequenciesin mammal cultured cells and the highest frequency was obtained when theratio was 1:9. The value 1:10 close to the above value was used at thistime).

(3) Analysis with PCR

Plasmid DNA was recovered from an embryo from which luminescence of GFPwas observed when the embryo became a tailbud. The recovery wasperformed by a treatment in which the embryo was placed in 100 μl of [10mM Tris-HCl, 10 mM EDTA (pH 8.0), 200 μg/ml proteinase K] and crushed,and subsequently kept at 50° C. for 12 hours or more. 2 μl out of thesolution was used as a template, and PCR was performed to detectexcision. The enzyme used was KOD Plus polymerase (Toyobo, Osaka,Japan). Primers were P1L (208th to 237th bases in GenBank file L09137)and P1R (770th to 741st bases), which correspond to a part of plasmidpUC19. A position where Tol1 element was incorporated was from the 400thto 441st bases, which was interposed between the primers. Concentrationsof dNTPs, MgSO₄ and the primers were respectively set to 0.2 mM, 2 mM,and 0.5 μM. Conditions of PCR are described in the correspondingsection.

(4) Cloning and Base Sequence Mapping

The reaction solution after completion of PCR was diluted with water to1/500, and the second PCR was performed using 1 μl out of the reactionsolution as a template. The primers used were P2L (338th to 367th basesof L01937) and P2R (650th to 621st bases). This nested type PCR is atreatment for facilitating cloning of the primary PCR product. Thesecondary PCR product was cloned at an EcoRV site of plasmid pBluescriptII KS(−) (Stratagene, La Jolla, Calif., USA) and base sequences wereexamined using a T3 primer and a T7 primer. ABI PRISM 310 GeneticAnalyzer (Applied Biosystems, Foster City, Calif., USA) was used for adevice.

2. Experimental Results

(1) Injection of Plasmid into Embryo and Recovery Thereof.

The experiment consists of two sets of A and B. In A, an indicator(plnd263GFP) and a complete helper (pHel851aa) were injected into anembryo of X. laevis from the two-cell to four-cell stages. B is acontrol section associated with functions of a transfer enzyme and adefective helper (pHel316aa) instead of a complete helper was injectedwith an indicator. DNA was injected in 154 embryos in A and 168 embryosin B, and respectively 112 embryos and 136 embryos survived untilbudtails. No obvious difference in survival rates was observed between Aand B (x²=3.07, DF=1, P>0.1).

Incorporation of injected DNA molecules in a nucleus is a preliminarycondition for realizing an experiment of excision detection. This isbecause only when transcription occurs in a nucleus and a transferenzyme gene carried on a helper plasmid is transcribed, the transferenzyme is supplied. In order to establish this premise, a GFP gene thatis carried on an indicator plasmid was utilized. When GFP is expressed,it means that a DNA type is incorporated into a nucleus. Ratios ofembryos in which GFP is expressed were 57% (64/112) in A and 65%(88/136) in B. There was no apparent difference between thesefrequencies (x²=1.48, DF=1, P>0.4). There was also no obvious differencein spacious patterns of GFP expression when the embryos were observedwith a microscope. 12 embryos having stronger GFP expression (A1 to A12)were selected from the embryos of A, and 12 embryos (B1 to B12) werealso selected from the embryos of B in the same manner, and plasmid DNAwas individually recovered from these 24 embryos in total.

(2) Analysis of Recovered Plasmids with PCR

Two modes of PCRs were performed using recovered DNA as a template. Theprimers used were P1L and P1R (placed in a position where Tol1 elementwas interposed between plnd263GFP).

The first PCR was to confirm that a DNA molecule of an indicator plasmidwas recovered. The distance between the two primers on plnd263GFP was2.4 kb, and a product having this length was confirmed in all samples(FIG. 22, upper column). Even though there was a difference in amountsof the products among the samples, there was no clear difference indegrees of fluctuation between A and B. It was thus confirmed that theindicator plasmid was recovered. If excision in a part from plnd263GFPto Tol1 element precisely occurred, 535 by of a fragment should havebeen amplified in PCR; however, no product with this size was found inall of the samples.

The other PCR was performed by shortening the time of an elongationreaction part (changed to 40 seconds from 150 seconds in the first time)(FIG. 22, lower column). The period of 40 seconds is not sufficient foramplifying the whole region of Tol1 element, and thus, it is expectedthat a product of excision can be efficiently amplified. When such atreatment was performed, a fragment having a size close to 535 by wasobserved in all samples of A (A1 to A12). In B, no fragment as a productwas observed. It can be inferred from the above-described results thatdropout of Tol1 element from plnd263GFP was caused in the embryos of Abut was not caused in the embryos of B.

(3) Analysis of Base Sequence of PCR Product

Base sequences of the PCR products obtained from 12 embryos of A wereanalyzed in order to examine positions and shapes of breaking pointsregarding DNA change generated in plnd263GFP. For preparation of theanalysis, first, the PCR products were again amplified by a nested typePCR. The primers used were P2L and P2R. Subsequently, fragments obtainedby this amplification were cloned to plasmids. At this time, only onecolony among the generated colonies was randomly selected for eachembryo. Therefore, it can be considered that 12 samples in this stagewere all generated by separate excisions. FIG. 23 shows the basesequences obtained from these 12 samples, which were aligned to check upeach other. Whole or most of sequences of Tol1 elements disappeared inall samples. It was revealed from the result that excision of Tol1element occurred during the time when an indicator was present in a frogcell. Whole regions of Tol1 element were dropped in 11 samples (A1 to A6and A8 to A 12) among the 12 samples, and 1 to 7 nucleotidescorresponding to a part of TSD were left. 39 nucleotides in the rightend of Tol1 element were left in sample A7, and 77 nucleotides in achromosome region adjacent to the left side of Tol1 disappeared. A partcorresponding to one TSD was included in these 77 nucleotides. It wasfurther found that new G residues were generated in 7 out of 12 samples.It can be considered from the finding that G was added or modificationto G occurred.

3. Discussion

An indicator (plnd256GFP) was injected into an embryo from two-cell tofour-cell stages with a complete helper (pHel851aa) or a defectivehelper (pHel316aa) in this study. There was no apparent difference inexpression frequencies and patterns of GFP in tailbud stages between Aand B. Therefore, it can be considered that there is no clear differencein incorporation efficiencies of DNA into nuclei between A and B.Additionally, amounts of indicators recovered from embryos were equalbetween A and B. In such situations, a clear difference in PCR to detectexcision was recognized between A and B. Accordingly, it can beconsidered that the cause of this difference is difference of basesequences of the two types of helpers. Only internal 6-nucleotideregions have differences. These portions are a codon for the 317th and318th amino acids in pHel851aa and are two termination codons inpHel316aa. The above description leads to the conclusion that dropout ofthe Tol1 portion from the indicator is due to catalysis by an enzymemade of pHel851aa and a protein made of pHel316aa does not have such anaction.

An important meaning of the above conclusion is that Tol1 element causesexcision in X. laevis cells. The fact that various traces accompanieddropout of Tol1 reinforces the meaning. This is because accompaniment oftraces to excision can be observed in many of DNA transposable elements.There are a large number of examples such as hobo of drosophila (Citeddocument 1), Activator of corn (Cited document 17), Tam3 of snapdragon(Cited document 4), mariner of drosophila (Cited document 2), and Tc1 ofnematode (Cited document 13).

It is an interesting phenomenon that G residues (C residues in the otherstrands) were generated in 7 samples. Since an analysis of basesequences was carried out in both strands, it is hard to consider thatthis phenomenon was generated by an artificial cause such as anexperimental method. Since the phenomena occurred in separate excisionsof no less than 7 samples, there is a possibility that some kinds of DNArepair mechanisms specific to this frog or general amphibians exist andthe phenomena reflect the mechanisms. Although the present inventorshave analyzed PCR products in 20 or more medaka fish and 20 or moremammal cultured cells so far, such a case has never been observed.

Excision is only a part of a transfer reaction of a DNA transposableelement. However, hAT family elements are considered to transfer in anunreplication mode, that is, a mode of inserting an excised fragmentitself in another site (Cited document 10), and the transfer reaction onthe whole in X. laevis cells will be surely realized.

Among examined 12 excisions, there was no sample in which a basesequence accurately returned to the original state. However, the factdoes not lead to the meaning that the dropped elements are alsoinaccurately incorporated into chromosomes. It is a phenomenonfrequently observed in a DNA transposable element that the element isprecisely cut out at an end and incorporated into a new place, butaddition or omission of nucleotides occurs in a hole after dropping. Thecauses are presumably that a reaction is ceased due to double-strandbreak repair (Cited document 13), nonhomologous recombination occurs(Cited document 16), and the like. The present inventors recentlyexamined two Tol1 elements newly inserted in mouse chromosomes bycloning. Then, such a result was obtained that the first to lastnucleotides of the element were exactly cloned with 8 bp of TSD.

DNA transposable elements such as Sleeping Beauty and Tol2 have beenused as tools for genomic manipulation in frogs (Cited documents 14 and5). However, Tol1 has a characteristic excelling these elements. Thecharacteristic is a point that Tol1 can carry a long DNA fragment (seeExample 2). Accordingly, Tol1 is not considered to be mere addition of agenetic manipulation technique intended for frogs, but should berecognized as a useful tool to open up a road to new development.

CITED DOCUMENTS

-   1. Atkinson, P. W., Warren, W. D. & O'Brochta, D. A. (1993). The    hobo transposable element of Drosophila can be cross-mobilized in    houseflies and excises like the Ac element of maize. Proceedings of    the National Academy of Sciences of the USA 90, 9693-9697.-   2. Bryan, G., Garza, D., Hartl, D. L. (1990). Insertion and excision    of the transposable element mariner in Drosophila. Genetics 125,    103-114.-   3. Cary, L. C., Goebel, M., Corsaro, B. G., Wang, H. G., Rosen, E.,    & Fraser, M. J. (1989). Transposon mutagenesis of baculoviruses:    analysis of Trichoplusiani transposon IFP2 insertions within the    FP-locus of nuclear polyhedrosis viruses. Virology 1 72, 156-169.-   4. Coen, E. S., Carpenter, R. & Martin, C. (1986). Transposable    elements generate novel spatial patterns of gene expression in    Antirrhinum majus. Cell 47, 285-296.-   5. Hamlet, M. R., Yergeau, D. A., Kuliyev, E., Takeda, M., Taira,    M., Kawakami, K. & Mead, P. E. (2006). Tol2 transposon-mediated    transgenesis in Xenopus tropicalis. Genesis 44, 438-445.-   6. Ivics, Z., Hackett, P. B., Plasterk, R. H. & Izsvak, Z. (1997).    Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon    from fish, and its transposition in human cells. Cell 91, 501-510.-   7. Koga, A., Shimada, A., Kuroki, T., Hori, H., Kusumi, J.,    Kyono-Hamaguchi, Y. & Hamaguchi, S. (2007). The Tol1 transposable    element of the medaka fish moves in human and mouse cells. Journal    of Human Genetics 52, 628-635.-   8. Koga, A., Suzuki, M., Inagaki, H., Bessho, Y. & Hori, H. (1996).    Transposable element in fish. Nature 383, 30.-   9. Koga, A., Inagaki, H., Bessho, Y. & Hori, H. (1995). Insertion of    a novel transposable element in the tyrosinase gene is responsible    for an albino mutation in the medaka fish, Oryzias latipes.    Molecular and General Genetics 249, 400-405.-   10. Kunze, R. (1996). The maize transposable element Activator (Ac).    In: H. Saedler and A. Gierl (ed.) Transposable Elements. Springer,    Berlin. pp. 161-194.-   11. Miskey, C., Izsvak, Z., Plasterk, R. H. & Ivics, Z. (2003). The    Frog Prince: a reconstructed transposon from Rana pipiens with high    transpositional activity in vertebrate cells. Nucleic Acids Research    31, 6873-6881.-   12. Peng, H. B. (1991). Appendix A. Solutions and protocols. In:    Kay, B. K. and Peng, H. B. (eds) Xenopus laevis: Practical Uses in    Cell and Molecular biology. Academic Press, San Diego, pp. 661-662.-   13. Plasterk, R. H. (1991). The origin of footprints of the Tc1    transposon of Caenorhabditis elegans. EMBO Journal 10, 1919-1925.-   14. Sinzelle, L., Vallin, J., Coen, L., Chesneau, A., Du Pasquier,    D., Pollet, N., Demeneix, B. & Mazabraud, A. (2006). Generation of    trangenic Xenopus laevis using the Sleeping Beauty transposon    system. Transgenic Research 15, 751-760.-   15. Tsutsumi, M., Imai, S., Kyono-Hamaguchi, Y., Hamaguchi, S.,    Koga, A. & Hori, H. (2006). Color reversion of the albino medaka    fish associated with spontaneous somatic excision of the Tol-1    transposable element from the tyrosinase gene. Pigment Cell Research    19, 243-247.-   16. Weinert, B. T., Min, B. & R10, D. C. (2005). P element excision    and repair by non-homologous end joining occurs in both G1 and G2 of    the cell cycle. DNA Repair 4, 171-181.-   17. Wessler, S. R., Baran, G., Varagona, M. & Dellaporta, S. L.    (1986). Excision of Ds produces waxy proteins with a range of    enzymatic activities. EMBO Journal 5, 2427-2432.-   18. Wu, S. C., Meir, Y. J., Coates, C. J., Handler, A. M., Pelczar,    P., Moisyadi, S. & Kaminski, J. M. (2006). piggyBac is a flexible    and highly active transposon as compared to Sleeping Beauty, Tol2,    and Mos1 in mammalian cells. Proceedings of the National Academy of    Sciences of the USA 103, 15008-15013.

Example 4

A transfer reaction can be regarded as one kind of DNA nonhomologousrecombination, and is considered to proceed in combination of actions ofendonuclease, polymerase, ligase, etc. It is still unclear at presentwhether a transfer enzyme has all of these functions. That is, it can beconsidered as a possibility that the transfer enzyme partly relies on ahost cell regarding required actions. When such a host factor isrequired, searching whether the factor is inherent to a host biologicalspecies or common in wide range of organisms is significant from theviewpoints of both biological evolution and biotechnology. In view ofevolution, this is because the meaning speculates at how much degree offrequencies transfer among species of transposable elements, that is,horizontal transfer is generated. It is necessary to regard horizontaltransfer as a factor that largely contributes to biological evolution ifit is frequent. From the viewpoint of biotechnology, a range of speciesto which developed gene introduction vectors, etc., are applicable isimportant. Among necessary host factors, fewer factors inherent tobiological species can be expected to have a wider applicable range. Itis speculated from the above-described results (Examples 1 to 3) thatTol1 element has a transposable activity in general vertebrates.

Higher animals diverge into two large phyletic lines in an early stageof evolution as shown in FIG. 24. The two phyletic lines are protostomesin which the blastopore generated in an initial developmental stage or aperiphery thereof becomes the mouse and deuterostomes in which theblastopore or a periphery thereof becomes the anus. Vertebrates belongto the latter. A trial was made to examine whether Tol1 elementtransfers even in the former or not by using an insect, silkworm.

1. Materials and Methods (1) Overview of Methods

The overall procedure is as shown in FIG. 25. DNA and RNA that were usedand details of each stage are described in the following.

(2) Transfer Enzyme RNA

Two types of plasmids (pTem851aa and pTem316aa) shown in FIG. 26 wereconstructed. Respective plasmids were used as templates and RNAs(mRNA851aa and mRNA316aa) were synthesized using RiboMAX Large Scale RNAProduction System (Promega Corp., Madison, Wis., USA). mRNA851aa encodesthe whole region of a Tol1 transfer enzyme made of 851 amino acids.mRNA316aa encodes from the initial to the 316th amino acids. The latterserves as a negative control associated with functions of a transferenzyme. These two types of RNA have the same whole lengths. Basesequences are different only in the part of 6 bases in the middle.

(3) Donor Plasmids

A plasmid shown in FIG. 27 was constructed. This is obtained by cloninga part of a tyrosinase gene of an albino medaka fish, and contains 1855by of Tol1 element. It is assumed that a transfer enzyme acts on asilkworm cell to cut out Tol1 element, and the Tol1 element transfersinto a chromosome of a silkworm. Here, 1855 by of this element does nothave a transfer enzyme gene (Cited document 5). Bacteria containing thedonor plasmid were grown in a liquid medium and a plasmid DNA was thenextracted and purified with the QIAGEN Plasmid Maxi Kit (QIAGEN GmbH,Hilden, Germany).

(4) Setting of Treatment Section

Three treatment sections (A, B and C) were set. Transposition issupposed to occur in A, and mRNA851aa was added to the donor plasmid andinjected into a silkworm fertilized egg. B is to confirm thattransposition does not occur if a transfer enzyme is incomplete, andmRNA316aa was added to the donor plasmid and injected into a silkwormfertilized egg. C is a negative control regarding a transpositiondetection method. Therefore, injection of DNA or RNA into a fertilizedegg was not carried out.

(5) Injection into Silkworm

In the treatment sections A and B, the donor plasmid and RNA were mixedso as to have final concentrations of 40 ng/μl and 160 ng/μl, andinjected into fertilized eggs within 40 minutes after being laid using aglass needle. Injection was performed on 250 fertilized eggs in A and 50fertilized eggs in B, and the fertilized eggs after completion ofinjection were stored in one plastic box. In the treatment section C, 50fertilized eggs were stored in the same plastic box without beingtreated with injection. Subsequently, the plastic box was kept warm at atemperature of 25° C. to promote development.

(6) Recovery of Plasmid DNA

After 5 to 6 hours from the start of warming, 75, 25 and 25 embryos wererespectively separated from the treatment sections A, B and C, and 25embryos each were placed in a centrifugal tube. Since three sets wereformed from A, these sets are determined to call A1, A2 and A3. Theremaining embryos were continued to be kept warm at 25° C. DNA wasextracted from the separated totally 5 groups of embryos by the Hirtmethod (Cited document 3). Cyclic DNA can be efficiently extracted bythis method.

(7) Detection of Excision

It was examined whether excision of Tol1 element occurred from a DNAmolecule of the recovered donor plasmid or not by PCR. The distancebetween primers Pex1 and Pex2 on the donor plasmid was 2.2 kb. Ifexcision of Tol1 element occurred while the donor plasmid was present ina silkworm cell, the distance between Pex1 and Pex2 became small in theDNA molecule. Therefore, when a product shorter than 2.2 kb wasgenerated in PCR, it is suggested that molecules in which excisionoccurred existed. When only a Tol1 element part was precisely drawn out,since Tol1 element had 1.9 kb, the size of the PCR product would be 0.3kb.

(8) Extraction of Genomic DNA

After 96 to 97 hours from the start of warming at 25° C., 100 embryoswere taken from the treatment section A, and genomic DNA was extracted.The extraction method was a standard method of treating with SDS andProteinase K, thereafter purifying DNA by salting out and an ethanolprecipitation method (Cited document 7). The obtained DNA was used fordetection of insertion. DNA extraction was performed not simultaneouslybut after detection of excision because it was expected that destructionof a donor plasmid would proceed as time passes. The donor plasmid has aregion corresponding to a PCR primer used in insertion detection.Therefore, a PCR product not derived from insertion would be generated.Accordingly, as destruction of the donor plasmid proceeds more, that is,as an embryo in a later stage is used, sensitivity of insertiondetection is expected to improve more.

(9) Detection of Insertion

Detection of insertion was performed in an inverse PCR technique. Tol1element contained in the donor plasmid does not have a breaking site bya restriction enzyme EcoRI. In the case where Tol1 element istransferred to a silkworm chromosome, when genomic DNA is cut withEcoRI, the chromosome regions are connected to both sides of Tol1element and a DNA fragment having the broken edges of EcoRI in the bothends is generated. When T4 DNA ligase acts on the DNA fragment, cyclicDNA in which both ends thereof are connected is generated. Primers Pin1and Pin2 are placed in both ends of Tol1 element and directed outwardthe Tol1 element. When PCR is carried out using these primers, in thecase where this cyclic DNA serves as a template, a PCR productcorresponding to the length of the contained chromosome is generated.The above-described sequential operations were performed to examinewhether Tol1 element was inserted into a chromosome or not.

(10) Cloning and Base Sequence Mapping

Necessary numbers of DNA molecules in the PCR product obtained inexcision detection and insertion detection were cloned to a plasmid andthe base sequence was mapped. The plasmid used for cloning was pT7Blue-2(Takara Bio Inc., Otsu, Japan). Synthesized single stranded DNAcorresponding to about 100 by of an upstream portion from the cloningpoint was used for a primer for base sequence mapping.

(11) PCR Conditions

PCR was heavily used in the above-described analysis. For a DNApolymerase, Ex Taq (Takara Bio Inc.) was used. Conditions such astemperature setting will be described in each section.

2. Results (1) Detection of Excision

DNA extracted from an embryo that was kept warm for 5 to 6 hours wasused as a template and primers (Pex1 and Pex2) located so as tointerpose Tol1 element therebetween on a donor plasmid was used toperform PCR. Results of electrophoresis after PCR is shown in FIG. 28.Elongation reactions in PCR were performed by setting two kinds oftimes; 150 seconds and 20 seconds. The time 150 seconds is a sufficientlength of time for amplifying 2.2 kb of a portion containing the wholeregion of Tol1 element on the donor plasmid. DNA fragments with 2.2 kbwere generated in the treatment sections A and B, but not observed inthe treatment section C. This result indicates that the DNA fragmentswith 2.2 kb were derived from the injected donor plasmid, not fromgenomic DNA of a silkworm. The results further showed that the donorplasmids were recovered in both of the treatment sections A and B.

PCR having a time for an elongation reaction of 20 seconds was carriedout in order to efficiently amplify a product from a DNA molecule inwhich excision occurred. In electrophoresis, DNA fragments appearedaround 0.3 kb in three lanes of the treatment section A. In thetreatment sections B and C, DNA fragments were not observed around thissize. These results suggested that excision of Tol1 element occurredonly in the treatment section A.

(2) Confirmation of Excision

Respective DNA fragments of A1, A2 and A3 were purified by an ethanolprecipitation method and then bonded to a plasmid vector to form clones.One clone was randomly selected from respective DNA fragments to examinea base sequence thereof. The results summarized so as to aligncorresponding portions are shown in FIG. 29. As understood from thedrawing, Tol1 element regions disappeared in all three clones. Further,a part of a region of target site duplication (TSD) on the both sideswas left. Then, 8 to 80 by of a fragment was added therebetween. A basesequence of newly added portion was checked with the whole base sequenceof Tol1 element, but no corresponding portion was found. The basesequence of TSD is CCTTTAGC, and a sequence complementary thereto isGCTAAAGG. In many of newly added portions, whole or a part of thiscomplementary sequence seems to be continuous.

It was clearly indicated form cloning and base sequence mapping of a PCRproduct that the whole region of Tol1 element disappeared. It was thusconfirmed that excision of Tol1 element from the donor plasmid occurred.

(3) Detection and Confirmation of Insertion

Genomic DNA was extracted from an embryo in the treatment section A thatwas kept warm for 96 to 97 hours. The genomic DNA was subjected to theabove-described cutting and circularization operations, thereafterinverse PCR was performed to clone the product to a plasmid. Whenseveral tens of clones were obtained as colonies of bacteria, twocolonies were randomly selected. Plasmid DNA was extracted and basesequence mapping was performed. The results summarized so as to aligncorresponding portions are shown in FIG. 30.

In three samples, base sequences of Tol1 element portions were conformedand portions outside Tol1 element were different. When only the sequenceof this portion was taken out from a clone of a silkworm and checkedwith the base sequence database of silkworms (KAIOKOBLAST;http://kaikoblast.dna.affrc.go.jp/), it was shown that a sequence having90% or more homology was present in a silkworm. In addition, TSD wasassumed not formed.

It was clearly indicated from the analysis of a base sequence of aninverse PCR product that a Tol1 element portion was connected to achromosome of a silkworm. It was thus confirmed that insertion of Tol1element occurred in a silkworm.

3. Discussion

Higher animals diverge to two large phyletic lines of protostomes anddeuterostomes at an early stage of evolution. Vertebrates such as humansand medaka fish belong to deuterostomes. Tol1 element is a DNAtransposable element present in a genome of medaka fish, and speculatedto have a transposable activity in general vertebrates. This studyexamined whether the Tol1 element transfers also in protostomes or notusing a silkworm as a material. The result clearly showed thattransposition occurred.

An enzyme catalyzing transposition of Tol1 element is a transfer enzymeof Tol1 element. However, a question if a transfer reaction proceedsonly with this enzyme or some factors derived from a host animal arenecessary has not been answered yet. The result obtained at this timethat “Tol1 element that transfers in deuterostomes such as humans andmedaka fish also transfers in a silkworm that is a protostome” gaveinformation to approach an answer regarding factors from a hostorganism. That is, obtained was the finding that “factors from a hostorganism are not necessary in a transfer reaction of Tol1 element;alternatively, even if the factors are necessary, they are common inprotostomes and deuterostomes.”

The result at this time has a significant meaning also in the field ofbiotechnology. First, the fact that Tol1 element transfers in a silkwormmeans that “systems such as gene introduction, gene trapping, andmutagenesis, which can be utilized in a silkworm, can be constructedusing Tol1 element.” Further, it can be expected that “systems developedusing Tol1 element have properties excelling those of systems that havebeen developed so far” as described below.

In a silkworm, a system using piggyBac element (Cited document 9) and asystem using Minos element (Cited document 11) have been developed sofar. Both of these two elements are elements belonging to themariner/Tc1 family. The mariner/Tc1 family is a group of transposableelements distributed in a wide range of organisms and has commonalityand similarity in structures and transfer mechanisms. The group wasnamed regarding mariner element of drosophila and Tc1 element ofnematode as typical examples. There is another large group oftransposable elements in addition to this family. The group is calledthe hAT family, and includes hobo element of drosophila, Activatorelement of corn, and Tam3 element of snapdragon (Cited document 1). Tol1element of medaka fish, which was proved to transfer in a silkworm atthis time, is an element belonging to the hAT family (Cited document 6).Characteristics of the hAT family when compared with those of themariner/Tc1 family include a point that “an element transfers even whenthe whole length is long.” For example, it was shown that when SleepingBeauty element that is a mariner/Tc1 family element has a whole lengthexceeding 9.1 kb, a transposable activity is almost lost in anexperiment using a cultured cell of a mouse (Cited document 12). On thecontrary, Tol1 element transfers in a cultured cell of a mouse at a highfrequency even when the Tol1 element has a whole length of 22.1 kb(Cited document 4). From this fact, utilization as a vector forintroduction of a long DNA fragment into a chromosome is particularlystrongly expected. Industrially useful genes with large whole lengthssuch as a fibroin gene have been known in a silkworm and its relatedspecies (Cited document 8). When such a gene is treated, Tol1 element isexpected to be an important vector.

Significance of the result at this time in the field of biotechnology isnot held only in a silkworm. The reason is that it is hard to considerthat only a silkworm among protostomes is in a particular situationregarding transposition of Tol1 element. It can be expected from theresults at this time that “Tol1 element will transfer also in manyprotostomes in addition to a silkworm.” It can be expected that systemssuch as gene introduction, gene trapping, and mutagenesis, which aredeveloped with Tol1 element, can be applied to a wide range oforganisms.

CITED DOCUMENTS

-   1. Calvi B. R., Hong T. J., Findley S. D., Gelbart W. M. (1991).    Evidence for a common evolutionary origin of inverted repeat    transposons in Drosophila and plants: hobo, Activator, and Tam3.    Cell 66: 465-471.-   2. Hikosaka A., Koga A. (2007). PCR detection of excision suggests    mobility of the medaka fish Tol1 transposable element in the frog    Xenopus laevis. Genet. Res.: in press.-   3. Hirt B. (1967). Selective extraction of polyoma DNA from infected    mouse cell cultures. J. Mol. Biol. 26: 365-369.-   4. Koga A., Higashide I., Hori H., Wakamatsu Y., Kyono-Hamaguchi Y.,    Hamaguchi S. (2007b). The Tol1 element of medaka fish is transposed    with only terminal regions and can deliver large DNA fragments into    the chromosomes. J. Hum. Genet. 52: 1026-1030.-   5. Koga A., Inagaki H., Bessho Y., Hori H. (1995). Insertion of a    novel transposable element in the tyrosinase gene is responsible for    an albino mutation in the medaka fish, Oryzias latipes. Mol. Gen.    Genet. 249: 400-405.-   6. Koga A., Shimada A., Kuroki T., Hori H., Kusumi J.,    Kyono-Hamaguchi Y., Hamaguchi S. (2007a). The Tol1 transposable    element of the medaka fish moves in human and mouse cells. J. Hum.    Genet. 52: 628-635.-   7. Sambrook J., Russell D. W. (2001) Molecular Cloning: A Laboratory    Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring    Harbor-   8. Sezutsu H., Yukuhiro K. (2000). Dynamic rearrangement within the    Antheraea pernyi silk fibroin gene is associated with four types of    repetitive units. J. Mol. Evol. 51: 329-338.-   9. Tamura T., Thibert C., Royer C., Kanda T., Abraham E., Kamba M.,    Komoto N., Thomas J. L., Mauchamp B., Chavancy G., Shirk P., Fraser    M., Prudhomme J. C., Couble P. (2000).-   10. Germline transformation of the silkworm Bombyx mori L. using a    piggyBac transposon-derived vector. Nat. Biotechnol. 18: 81-84.-   11. Uchino K., Imamura M., Shimizu K., Kanda T., Tamura T. (2007).    Germ line transformation of the silkworm, Bombyx mori, using the    transposable element Minos. Mol. Genet. Genomics 277: 213-220.-   12. Karsi A., Moav B., Hackett P., Liu Z. (2001) Effects of insert    size on transposition efficiency of the Sleeping Beauty transposon    in mouse cells. Mar Biotechnol 3: 241-245.

INDUSTRIAL APPLICABILITY

The present invention provides a Tol1 element transposase, a DNAintroduction system using the same, and the like. The present inventionis intended for applications such as gene introduction, gene targeting,mutagenesis, trapping of genes, promoters, enhancers, etc.

The present invention is not limited to the description of the aboveembodiments and examples of the invention. Various modified forms withinthe range where a skilled person can easily conceive of are alsoincluded in the invention without departing from the description of thescope of claims for the patent.

Entire contents of treatises, unexamined patent publications, patentpublications, and the like, indicated in the present specification arehereby incorporated by reference.

1. A Tol1 element transposase comprising any of proteins selected fromthe group consisting of the following (a) to (c): (a) a protein havingan amino acid sequence encoded by the base sequence of SEQ ID NO: 1; (b)a protein having the amino acid sequence of SEQ ID NO: 2; and (c) aprotein having an amino acid sequence homologous to the amino acidsequence of SEQ ID NO: 2, and having an enzymatic activity fortransferring Tol1 element.
 2. A polynucleotide encoding a Tol1 elementtransposase comprising any of base sequences selected from the groupconsisting of the following (a) to (c): (a) a base sequence encoding theamino acid sequence of SEQ ID NO: 2; (b) the base sequence of SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO: 4; and (c) a base sequence homologous tothe base sequence (b) and encoding a protein having an enzymaticactivity for transferring Tol1 element.
 3. An expression constructcomprising the polynucleotide according to claim
 2. 4. The expressionconstruct according to claim 3, further comprising a promoter operablylinked to the polynucleotide.
 5. The expression construct according toclaim 3, further comprising a poly-A additional signal sequence or apoly-A sequence connected to the polynucleotide in the downstream side.6. A DNA introduction system comprising: (a) a donor factor having sucha structure that desired DNA is inserted in a transposase gene-defectedTol1 element; and (b) a helper factor containing the transposaseaccording to claim
 1. 7. The DNA introduction system according to claim6, wherein the Tol1 element has the inverted repeat sequence of SEQ IDNO: 5 in the 5′ end region and the inverted repeat sequence of SEQ IDNO: 6 in the 3′ end region.
 8. The DNA introduction system according toclaim 6, wherein the Tol1 element comprises DNA of the following (a) or(b): (a) DNA having the base sequence of any of SEQ ID NOs: 10 to 12; or(b) DNA having a base sequence homologous to the base sequence of any ofSEQ ID NOs: 10 to 12, wherein a transposase having the amino acidsequence of SEQ ID NO: 1 binds to its end.
 9. The DNA introductionsystem according to claim 6, wherein the Tol1 element comprises 5′ endside DNA and 3′ end side DNA obtained by deleting at least from the158th base to the 1749th base counting from the 5′ end in the basesequence of SEQ ID NO:
 10. 10. The DNA introduction system according toclaim 6, wherein the Tol1 element comprises DNA having the base sequenceof SEQ ID NO: 21 and DNA having the base sequence of SEQ ID NO:
 22. 11.The DNA introduction system according to claim 8, wherein a target siteduplicated sequence is connected to the 5′ end and the 3′ end of thetoll element.
 12. The DNA introduction system according to claim 11,wherein the target site duplicated sequence comprises the sequence ofany of SEQ ID NOs: 13 to
 15. 13. The DNA introduction system accordingto claim 6, wherein the desired DNA is a gene.
 14. The DNA introductionsystem according to claim 6, wherein the donor factor is a vectorobtained by inserting a desired DNA in a transposase gene-defected Tol1element, and the helper factor is a vector comprising the polynucleotideaccording to claim
 2. 15. The DNA introduction system according to claim14, wherein the vector being the helper factor further comprises apromoter operably linked to the polynucleotide.
 16. The DNA introductionsystem according to claim 14, wherein the vector being the helper factorfurther comprises a poly-A additional signal sequence or a poly-Asequence connected to the polynucleotide in the downstream side.
 17. ADNA introduction method comprising a step of introducing the DNAintroduction system according to claim 6 to a target cell which is avertebrate cell.
 18. The DNA introduction method according to claim 17,wherein the target cell is a vertebrate cell other than a cell in astate of a constituent factor of a human individual.
 19. The DNAintroduction method according to claim 17, further comprising a step ofintroducing DNA different from the desired DNA introduced by the DNAintroduction system to the target cell by utilizing Tol2 element.
 20. Amethod of transferring a specific DNA site on genomic DNA, comprising astep of supplying a transposase corresponding to Tol1 element or Tol2element to a cell genetically manipulated with the DNA introductionmethod according to claim
 19. 21. A method of transferring a specificDNA site on genomic DNA, comprising a step of introducing thetransposase according to claim 1 into a cell having a transposasegene-defected Tol1 element on genomic DNA.
 22. The method according toclaim 21, wherein another polynucleotide sequence is inserted in theTol1 element.
 23. A cell genetically manipulated by the DNA introductionsystem according to claim
 6. 24. A DNA introducing kit, comprising: adonor factor comprising an expression construct containing a transposasegene-defected Tol1 element and having an insertion site; and a helperfactor comprising an expression construct containing the transposaseaccording to claim
 1. 25. The DNA introducing kit according to claim 24,wherein the toll element comprises a structure having the insertion sitebetween 5′ end side DNA and 3′ end side DNA, which is obtained bydeleting at least from the 158th base to the 1749th base counting fromthe 5′ end in the base sequence of SEQ ID NO:
 10. 26. The DNAintroducing kit according to claim 24, wherein the Tol1 elementcomprises a structure having the insertion site between DNA having thebase sequence of SEQ ID NO: 21 and DNA having the base sequence of SEQID NO:
 22. 27. The DNA introducing kit according to claim 24, whereinthe insertion site is made of a plurality of different kinds ofrestriction enzyme recognition sites.
 28. The DNA introducing kitaccording to claim 24, wherein the donor factor is a vector comprising atransposase gene-defected Tol1 element and an insertion site, and thehelper factor is a vector comprising the polynucleotide according toclaim
 2. 29. The DNA introducing kit according to claim 28, wherein thevector being the helper factor further comprises a promoter operablylinked to the polynucleotide.
 30. The DNA introducing kit according toclaim 28, wherein the vector being the helper factor further comprises apoly-A additional signal sequence or a poly-A sequence connected to thepolynucleotide in the downstream side.
 31. A reconstructed transposonhaving a structure inserted with the polynucleotide according to claim 2in a transposase gene-defected Tol1 element.
 32. The transposonaccording to claim 31, comprising a promoter operably linked to thepolynucleotide.
 33. The transposon according to claim 31, comprising apoly-A additional signal sequence or a poly-A sequence connected to thepolynucleotide in the downstream side.
 34. A DNA introduction system,comprising the transposon according to claim 31.